U.S. patent application number 09/827822 was filed with the patent office on 2002-07-11 for peptides that block viral infectivity and methods of use thereof.
Invention is credited to Vahlne, Anders.
Application Number | 20020091086 09/827822 |
Document ID | / |
Family ID | 23459335 |
Filed Date | 2002-07-11 |
United States Patent
Application |
20020091086 |
Kind Code |
A1 |
Vahlne, Anders |
July 11, 2002 |
Peptides that block viral infectivity and methods of use
thereof
Abstract
The discovery of peptides in amide form that inhibit viral
infection, including human immunodeficiency virus (HIV) infection
is disclosed. Methods of use of peptides are also disclosed
including use in medicaments for the treatment and prevention of
viral infection, such as HIV infection.
Inventors: |
Vahlne, Anders; (Hovas,
SE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
23459335 |
Appl. No.: |
09/827822 |
Filed: |
April 6, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09827822 |
Apr 6, 2001 |
|
|
|
09370368 |
Aug 9, 1999 |
|
|
|
Current U.S.
Class: |
435/235.1 ;
435/5; 514/21.9; 514/3.8; 530/300; 530/331 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 31/00 20180101; A61P 31/18 20180101; A61P 31/12 20180101; Y10S
977/916 20130101; C12N 2740/15022 20130101; C12N 2740/16122
20130101; C07K 14/005 20130101 |
Class at
Publication: |
514/18 ; 514/2;
435/5; 435/235.1; 530/300; 530/331 |
International
Class: |
A01N 037/18; C12Q
001/70; C12N 007/01; C07K 004/00; C07K 007/00; C07K 016/00; A61K
038/06; A61K 038/00; C12N 007/00; C07K 002/00; C07K 005/00; C07K
014/00; C07K 017/00 |
Claims
What is claimed is:
1. A method of inhibiting propagation of a virus in a host cell
comprising: providing said cell with an effective amount of a
tripeptide in amide form consisting of the formula
X.sub.1X.sub.2X.sub.3-NH.sub.2, wherein X.sub.1, X.sub.2, and
X.sub.3 are any amino acid, wherein said tripeptide is not
Gly-Pro-Gly-NH.sub.2, and wherein said tripeptide has a sequence
that corresponds to a sequence of a capsid protein of said
virus.
2. The method of claim 1, wherein said virus is a retrovirus.
3. The method of claim 1, wherein said virus is a lentivirus.
4. The method of claim 3, wherein said virus is HIV.
5. The method of claim 4, wherein said tripeptide is
Gly-Lys-Gly-NH.sub.2
6. The method of claim 4, wherein said tripeptide is
Arg-Gln-Gly-NH.sub.2
7. The method of claim 4, wherein said tripeptide is
Cys-Gln-Gly-NH.sub.2.
8. The method of claim 4, wherein said tripeptide is
Lys-Gln-Gly-NH.sub.2
9. The method of claim 4, wherein said tripeptide is
Ala-Leu-Gly-NH.sub.2.
10. The method of claim 4, wherein said tripeptide is
Gly-Val-Gly-NH.sub.2
11. The method of claim 4, wherein said tripeptide is
Val-Gly-Gly-NH.sub.2
12. The method of claim 4, wherein said tripeptide is
Ala-Ser-Gly-NH.sub.2
13. The method of claim 4, wherein said tripeptide is
Ser-Leu-Gly-NH.sub.2
14. The method of claim 4, wherein said tripeptide is
Ser-Pro-Thr-NH.sub.2.
15. The method of claim 1, further compri sin g providing to said
cell an antiviral treatment selected from the group consisting of
nucleoside analogue reverse transcriptase inhibitors, nucleotide
analogue reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors and protease inhibitors.
16. The method of claim 1, wherein said tripeptide is joined to a
support.
17. The method of claim 1, wherein said tripeptide is provid ed to
said cell in combination with a pharmaceutically acceptable
carrier.
18. A method of identifying a tripeptide in amide form for
incorporation into an anti-viral pharmaceutical comprising: (a)
selecting a tripeptide sequence that corresponds to a capsid
sequence of a virus; (b) providing a tripeptide in amide form
consisting of said tripeptide; (c) contacting a plurality of cells
infected with said virus with an effective amount of said
tripeptide; and (d) determining whether said contacting results in
inhibition of propagation of said virus.
19. The method of claim 18, wherein said virus is a retrovirus.
20. The method of claim 18, wherein said virus is a lentivirus.
21. The method of claim 18, wherein said virus is HIV.
22. A method of inhibiting propagation of HIV in a human
comprising: identifying a human in need of a medicament that
inhibits propagation of HIV; and providing to said human an
effective amount of a tripeptide in amide form consisting of the
formula X.sub.1X.sub.2X.sub.3-NH.sub.2, wherein X.sub.1, X.sub.2,
and X.sub.3 are any amino acid, wherein said tripeptide is not
Gly-Pro-Gly-NH.sub.2, and wherein said tripeptide has a sequence
that corresponds to a sequence of p24.
23. The method of claim 22, wherein said tripeptide is
Gly-Lys-Gly-NH.sub.2
24. The method of claim 22, wherein said tripeptide is
Arg-Gln-Gly-NH.sub.2
25. The method of claim 22, wherein said tripeptide is
Cys-Gln-Gly-NH.sub.2.
26. The method of claim 22, wherein said tripeptide is
Lys-Gln-Gly-NH.sub.2
27. The method of claim 22, wherein said tripeptide is
Ala-Leu-Gly-NH.sub.2.
28. The method of claim 22, wherein said tripeptide is
Gly-Val-Gly-NH.sub.2.
29. The method of claim 22, wherein said tripeptide is
Val-Gly-Gly-NH.sub.2.
30. The method of claim 22, wherein said tripeptide is
Ala-Ser-Gly-NH.sub.2.
31. The method of claim 22, wherein said tripeptide is
Ser-Leu-Gly-NH.sub.2.
32. The method of claim 22, wherein said tripeptide is
Ser-Pro-Thr-NH.sub.2.
33. The method of claim 22, further comprising providing to said
human an antiviral treatment selected from the group consisting of
nucleoside analogue reverse transcriptase inhibitors, nucleotide
analogue reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors and protease inhibitors.
34. The method of claim 22, wherein said tripeptide is joined to a
support.
35. The method of claim 22, wherein said tripeptide is provided to
said human in combination with a pharmaceutically acceptable
carrier.
36. The method of claim 22, wherein said tripeptide is provided to
said human topically.
37. The method of claim 22, wherein said tripeptide is provided to
said human transdermally.
38. The method of claim 22, wherein said tripeptide is provided to
said human parenterally.
39. The method of claim 22, wherein said tripeptide is provided to
said human gastrointestinally.
40. The method of claim 22, wherein said tripeptide is provided to
said human transbronchially.
41. The method of claim 22, wherein said tripeptide is provided to
said human transalveolarly.
Description
RELATED APPLICATIONS
[0001] This application is a divisional that claims priority to
application Ser. No. 09/370,368, filed Aug. 9, 1999, which is
hereby expressly incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention is related to the discovery of
peptides that inhibit viral infection, including human
immunodeficiency virus (HIV) infection. More specifically,
medicaments comprising various small peptides are disclosed for use
in the treatment and prevention of viral infection, such as HIV
infection.
BACKGROUND OF THE INVENTION
[0003] All viruses are composed of a protein shell surrounding a
nucleic acid containing core. The protein shell directly
surrounding the viral nucleic acid is called a capsid, whereas, the
complete protein-nucleic acid complex having both the capsid and
the nucleic acid is called a nucleocapsid. Arenaviruses,
rotaviruses, orbiviruses, retroviruses (including lentiviruses),
papillomaviruses, adenoviruses, herpesviruses, paramyxovirus,
myxovirus, and hepadnaviruses all exhibit these general structural
features. (Virology, Fields ed., third edition, Lippencott-Raven
publishers, pp 1513, 1645, 1778, 2047, 2113, 2221, and 2717
(1996)).
[0004] The capsid is composed of many subunits (capsomeres) and
capsomeres are formed from several homo or hetero-polymers of
protein. The noncovalent bonds between capsomeres in a viral
assembly are of the same sort that stabilize a folded protein
domain. The interface between two subunits can look very much like
a single domain, with amino acid side chains tightly packed against
one another. A common feature to most of the virus structures
analyzed is the way in which a polypeptide chain from one capsomere
can extend under or over domains of neighboring capsomeres. These
extended polypeptide arms intertwine with other polypeptide arms
and help to stabilize the capsid by initiating hydrophobic
interactions, hydrogen bonding, and salt bridges. Contacts between
individual capsomeres, and for some viruses also contacts with core
proteins, determine the overall capsid structure and if a number of
identical capsomeres are involved, repeated contacts occur and the
resulting structure is symmetrical. (Virology, Fields ed., third
edition, Lippencott-Raven publishers, p 62 (1996)).
[0005] Some simple viruses form spontaneously from their
dissociated components while others require enzyme-catalyzed
modifications of the capsomeres to trigger assembly. Viral self
assembly is driven by the stability of the interactions between
protein subunits under conditions that favor association. More
complex viruses are often constructed from subassemblies that have
undergone self assembly processes. (Virology, Fields ed., third
edition, Lippencott-Raven publishers, pp 62, 70, 1646 and 1888
(1996)). Although the capsids of many viruses differ in protein
composition, a general viral structural design has evolved
characterized by polymerized capsomeres that, in turn, are composed
of several homo- or hetero- polymers of protein.
[0006] HIV is the name given to a lentivirus that infects humans
and that causes acquired immuno-deficiency syndrome (AIDS). The
lentivirus isolates from humans are grouped into one of two types
(HIV-1 and HIV-2) on the basis of serologic properties and sequence
analysis of molecularly cloned viral genomes. Genetically distinct
lentiviruses have been obtained from several non-human primate
species including African green monkeys, sooty magabeys, mandrills,
chimpanzees, and sykes. Collectively, the lentivirus isolates from
non-human primates are called SIV. Sequence analysis reveals that
the genomes of some SIV strains and HIV-1 and HIV-2 strains exhibit
a high degree of homology. Further, electron microscopy reveals
that the ultrastructure of HIV and SIV are similar in that both
have virions about 110 nm in diameter with a cone-shaped
nucleocapsid surrounded by a lipid bilayer membrane that contains
envelope glycoprotein spikes. (Virology, Fields ed., third edition,
Lippencott-Raven publishers, pp 1882-1883 (1996)).
[0007] HIV is a complex retrovirus containing at least seven genes.
The viral structural genes, designated gag, pol, and env,
respectively code for the viral core proteins, reverse
transcriptase, and the viral glycoproteins of the viral envelope.
The remaining HIV genes are accessory genes involved in viral
replication. The gag and env genes encode polyproteins, i.e., the
proteins synthesized from each of these genes are
post-translationally cleaved into several smaller proteins.
[0008] Although the overall shape of HIV and SIV virions is
spherical, the nucleocapsid is asymmetrical having a long dimension
of about 100 nm, a wide free end about 40-60 nm, and a narrow end
about 20 nm in width. The nucleocapsid within each mature virion is
composed of two molecules of the viral single-stranded RNA genome
encapsulated by proteins proteolytically processed from the Gag
precursor polypeptide. Cleavage of the gag gene polyprotein
Pr55.sup.gag by a viral coded protease (PR) produces mature capsid
proteins. These gag gene products are the matrix protein (p17),
that is thought to be located between the nucleocapsid and the
virion envelope; the major capsid protein (p24), that forms the
capsid shell; and the nucleocapsid protein (p9), that binds to the
viral RNA genome. This proteolytic processing in infected cells is
linked to virion morphogenesis. (Virology, Fields ed., third
edition, Lippencott-Raven publishers, pp 1886-1887 (1996)).
[0009] The major capsid protein p24 (also called CA) contains about
240 amino acids and exhibits a molecular weight of 24-27 kD. The
protein p24 self-associates to form dimers and oligomeric complexes
as large as dodecamers. Genetic studies with mutations in the HIV-1
gag polyprotein have identified several functional domains in the
p24 protein including the C terminal half of the molecule and a
major homology region (MHR) spanning 20 amino acids that is
conserved in the p24 proteins of diverse retroviruses. These
mutations appear to affect precursor nucleocapsid assembly.
(Virology, Fields ed., third edition, Lippencott-Raven publishers,
pp 1888-1889 (1996)).
[0010] Since the discovery of HIV-1 as the etiologic agent of AIDS,
significant progress has been made in understanding the mechanisms
by which the virus causes disease. While many diagnostic tests have
been developed, progress in HIV vaccine therapy has been slow
largely due to the heterogeneous nature of the virus and the lack
of suitable animal models. (See, e.g., Martin, Nature, 345:572-573
(1990)).
[0011] A variety of pharmaceutical agents have been used in
attempts to treat AIDS. Many, if not all, of these drugs, however,
create serious side effects that greatly limit their usefulness as
therapeutic agents. HIV reverse transcriptase is one drug target
because of its crucial role in viral replication. Several
nucleoside derivatives have been found to inhibit HIV reverse
transcriptase including azidothymidine (AZT, zidovidine.RTM.). AZT
causes serious side effects such that many patients cannot tolerate
its administration. Other nucleoside analogs that inhibit HIV
reverse transcriptase have been found to cause greater side effects
than AZT. Another drug target is the HIV protease (PR) crucial to
virus development. PR is an aspartic protease and can be inhibited
by synthetic compounds. (Richards, FEBS Lett., 253:214-216 (1989)).
Protease inhibitors inhibit the growth of HIV more effectively than
reverse transcriptase inhibitors but prolonged therapy has been
associated with metabolic diseases such as lipodystrophy,
hyperlipidemia, and insulin resistance.
[0012] Additionally, HIV quickly develops resistance to
nucleoside/nucleotide analogue reverse transcriptase inhibitors and
protease inhibitors. This resistance can also spread between
patients. Studies have shown, for example, that one tenth of the
individuals recently infected by HIV already have developed
resistance to AZT, probably because they were infected by a person
that at the time of transmission carried a virus that was resistant
to AZT.
[0013] It would be useful in the treatment and prevention of viral
infections, including HIV and SIV, to have specific and selective
therapeutic agents that cause few, if any, side effects.
SUMMARY OF THE INVENTION
[0014] The present invention is related to small peptides (two to
ten amino acids in length) that inhibit viral infectivity. An
intact capsid structure is of vital importance for the infectivity
of a virion. A way to disrupt assembly of capsid protein
macromolecules, that for their infectivity are dependent on di-,
tri-, tetra-, or polymerization, is to construct small molecules
that affect such protein-protein interactions. It was discovered
that small peptides with their carboxyl terminus hydroxyl group
replaced with an amide group have such an inhibiting effect on
capsid-protein interactions. Thus, aspects of the present invention
relate to modified small peptides that effect viral capsid
assembly.
[0015] In desirable embodiments, the short peptides bind to a
protein that is involved in capsomere organization and capsid
assembly of HIV-1, HIV-2, and SIV and thereby inhibit and/or
prevent proper capsid assembly and, thus, viral infection. The
small peptides Gly-Pro-Gly-NH.sub.2 (GPG-NH.sub.2),
Gly-Lys-Gly-NH.sub.2 (GKG-NH.sub.2), Cys-Gln-Gly-NH.sub.2
(CQG-NH.sub.2), Arg-Gln-Gly-NH.sub.2 (RQG-NH.sub.2),
Lys-Gln-Gly-NH.sub.2 (KQG-NH.sub.2), Ala-Len-Gly-NH.sub.2
(ALG-NH.sub.2), Gly-Val-Gly-NH.sub.2 (GVG-NH.sub.2),
Val-Gly-Gly-NH.sub.2 (VGG-NH.sub.2), Ala-Ser-Gly-NH.sub.2
(ASG-NH.sub.2), Ser-Leu-Gly-NH.sub.2 (SLG-NH.sub.2), and
Ser-Pro-Thr-NH.sub.2 (SPT-NH.sub.2) are the preferred species.
These small peptides and peptidomimetics resembling their structure
(collectively referred to as "peptide agents") are used in a
monomeric or multimeric form. The peptide agents of the present
invention are suitable for therapeutic and prophylactic application
in mammals, including man, suffering from viral infection.
[0016] In one embodiment, a composition for inhibiting viral
replication in host cells infected with a virus has an effective
amount of a peptide in amide form having the formula
X.sub.1X.sub.2X.sub.3-NH.sub.2, wherein X.sub.1, X.sub.2, and
X.sub.3 are any amino acid and said peptide is not
Gly-Pro-Gly-NH.sub.2, and wherein said composition inhibits viral
replication by interrupting viral capsid assembly. Desirably,
X.sub.3 of these peptides is glycine. Additionally, the
compositions described above can include a peptide in amide form
selected from the group consisting of peptides having the formula
Gly-Lys-Gly-NH.sub.2, Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2,
Lys-Gln-Gly-NH.sub.2, Ala-Leu-Gly-NH.sub.2, Gly-Val-Gly-NH.sub.2,
Val-Gly-Gly-NH.sub.2, Ala-Ser-Gly-NH.sub.2, Ser-Leu-Gly-NH.sub.2,
and Ser-Pro-Thr-NH.sub.2.
[0017] In another related embodiment, the composition described
above is a peptide in amide form that has the formula
X.sub.4X.sub.5X.sub.1X.sub.2X.- sub.3-NH.sub.2, wherein X.sub.4 and
X.sub.5 are any amino acid and any one or two amino acids can be
absent. This embodiment can be a tripeptide having the formula
X.sub.1X.sub.2X.sub.3, wherein the sequence is found in the amino
acid sequence of the capsid protein of the virus.
[0018] In some embodiments, the compositions described above are
joined to a support and in other embodiments, the compositions
described above are incorporated into a pharmaceutical having a
pharmaceutically acceptable carrier. For example, the peptide in
amide form can have the formula Gly-Lys-Gly-NH.sub.2. and can be
joined to a support. Further, the peptide in amide form can have a
formula such as Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2,
Lys-Gln-Gly- NH.sub.2, Ala-Leu-Gly- NH.sub.2, or Ser-Leu-Gly-
NH.sub.2. Thes peptides can also be joined to a support.
[0019] Methods of inhibiting HIV replication in a host cell are
also embodiments. One approach, for example, involves administering
to a cell an effective amount of a peptide in amide form having the
formula X.sub.1X.sub.2X.sub.3-NH.sub.2, wherein X.sub.1, X.sub.2,
and X.sub.3 are any amino acid and said peptide is not
Gly-Pro-Gly-NH.sub.2. Accordingly, the peptide above can be
selected from the group consisting of peptides having the formula
Gly-Lys-Gly-NH.sub.2, Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2,
Lys-Gln-Gly-NH.sub.2, Ala-Leu-Gly-NH.sub.2, Gly-Val-Gly-NH.sub.2,
Val-Gly-Gly-NH.sub.2, Ala-Ser-Gly-NH.sub.2, Ser-Leu-Gly-NH.sub.2,
and Ser-Pro-Thr-NH.sub.2. The method described above can further
include the step of administering an antiviral treatment selected
from the group consisting of nucleoside analogue reverse
transcriptase inhibitors, nucleotide analogue reverse transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors, and
protease inhibitors. The peptide used in the method above can
bejoined to a support or can be administered in a pharmaceutical
comprising a pharmaceutically acceptable carrier.
[0020] In another embodiment, a composition for inhibiting HIV
replication in host cells includes an effective amount of a peptide
in amide form having the formula X.sub.1X.sub.2X.sub.3-NH.sub.2,
wherein X.sub.1, X.sub.2, and X.sub.3 are any amino acid and said
peptide is not Gly-Pro-Gly-NH.sub.2 and wherein said composition
inhibits HIV replication by interrupting assembly of the capsid.
Desirably, X.sub.3 is glycine in the peptides of this embodiment.
Preferably, the peptide of this embodiment is selected from the
group consisting of peptides having the formula
Gly-Lys-Gly-NH.sub.2, Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2,
Lys-Gln-Gly-NH.sub.2, Ala-Leu-Gly-NH.sub.2, Gly-Val-Gly-NH.sub.2,
Val-Gly-Gly-NH.sub.2, Ala-Ser-Gly-NH.sub.2, Ser-Leu-Gly-NH.sub.2,
and Ser-Pro-Thr-NH.sub.2. Additionally, the peptide in amide form,
described above, can have the formula
X.sub.4X.sub.5X.sub.1X.sub.2X.sub.3-NH.sub.2, wherein X.sub.4 and
X.sub.5 are amino acids and wherein any one or two, amino acids is
absent. These compositions can have a tripeptide
X.sub.1X.sub.2X.sub.3 that is found in the amino acid sequence of
the capsid protein of HIV, for example. In some embodiments, these
peptides are joined to a support and in other embodiments, these
peptides are incorporated into a pharmaceutical comprising a
pharmaceutically acceptable carrier.
[0021] In another method, an approach to inhibit viral replication
in host cells is provided, which involves administering to said
cells an effective amount of a peptide in amide form having the
formula X.sub.1X.sub.2X.sub.3-NH.sub.2, wherein X.sub.1, X.sub.2,
and X.sub.3 are any amino acid and said peptide is not
Gly-Pro-Gly-NH.sub.2. In this method, the peptide can be selected
from the group consisting of peptides having the formula
Gly-Lys-Gly-NH.sub.2, Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2,
Lys-Gln-Gly-NH.sub.2, Ala-Leu-Gly-NH.sub.2, Gly-Val-Gly-NH.sub.2,
Val-Gly-Gly-NH.sub.2, Ala-Ser-Gly-NH.sub.2, Ser-Leu-Gly-NH.sub.2,
and Ser-Pro-Thr-NH.sub.2. This method can also include the step of
administering an antiviral treatment selected from the group
consisting of nucleoside analogue reverse transcriptase inhibitors,
nucleotide analogue reverse transcriptase inhibitors,
non-nucleoside reverse transcriptase inhibitors, and protease
inhibitors. Further, the peptide used in this method can be joined
to a support or can be administered in a pharmaceutical comprising
a pharmaceutically acceptable carrier.
[0022] In another method, an approach for interrupting viral capsid
assembly is provided. This approach involves contacting a cell with
an effective amount of a peptide in amide form having the formula
X.sub.1X.sub.2X.sub.3-NH.sub.2, wherein X.sub.1, X.sub.2, and
X.sub.3 are any amino acid and said peptide is not
Gly-Pro-Gly-NH.sub.2. Desirably, the peptide used in this method is
selected from the group consisting of peptides having the formula
Gly-Lys-Gly-NH.sub.2, Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2,
Lys-Gln-Gly-NH.sub.2, Ala-Leu-Gly-NH.sub.2, Gly-Val-Gly-NH.sub.2,
Val-Gly-Gly-NH.sub.2, Ala-Ser-Gly-NH.sub.2, Ser-Leu-Gly-NH.sub.2,
and Ser-Pro-Thr-NH.sub.2. In some embodiments, X.sub.3 is glycine
in the peptide used in this method. In other embodiments, the
method employs a peptide in amide form having the formula
X.sub.4X.sub.5X.sub.1X.sub.2X.sub.3-NH.sub.2 wherein X.sub.4 and
X.sub.5 are an amino acid and, wherein any one or two amino acids
is absent. Still further, the method can involve the use of a
tripeptide X.sub.1X.sub.2X.sub.3 that is found in the amino acid
sequence of a protein of the virus. Oftentimes the peptide of the
method is joined to a support or is incorporated in a
pharmaceutical.
[0023] Methods of identification of peptide agents are also
provided. By one method, for example, a peptide agent for
incorporation into a anti-viral pharmaceutical is identified by
contacting a plurality of cells infected with a virus with an
effective amount of a peptide agent, wherein said peptide is not
Gly-Pro-Gly-NH.sub.2, analyzing the virus for incomplete capsid
formation, and selecting the peptide agent that induces incomplete
capsid formation. This method can involve an analysis of capsid
formation that employs microscopy and the virus can be selected
from the group consisting of HIV-1, HIV-2, and SIV. Further, the
peptide agent identified can be selected from the group consisting
of a tripeptide, an oligopeptide, and a peptidomimetic. For
example, the peptide agent above can be selected from the group
consisting of peptides having the formula Gly-Lys-Gly-NH.sub.2,
Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2, Lys-Gln-Gly-NH.sub.2,
Ala-Leu-Gly-NH.sub.2, Gly-Val-Gly-NH.sub.2, Val-Gly-Gly-NH.sub.2,
Ala-Ser-Gly-NH.sub.2, Ser-Leu-Gly-NH.sub.2, and
Ser-Pro-Thr-NH.sub.2. In a preferred embodiment, the peptide agent
used in the method above has an amino acid sequence that
corresponds to an amino acid sequence of p24.
[0024] In another embodiment, a method for identifying a peptide
agent that binds to a viral protein is provided, which involves
providing a viral protein, contacting the viral protein with an
effective amount of a peptide agent, wherein said peptide agent is
not Gly-Pro-Gly-NH.sub.2, and detecting the formation of a complex
comprising the viral protein and the peptide agent. As above, this
method can involve the use of a viral protein that is from a virus
selected from the group consisting of HIV-1, HIV-2, and SIV.
Further, in some aspects, the peptide agent is selected from the
group consisting of a tripeptide, an oligopeptide, and a
peptidomimetic. Desirably, the method above employs a peptide agent
is selected from the group consisting of peptides having the
formula Gly-Lys-Gly-NH.sub.2, Arg-Gln-Gly-NH.sub.2,
Cys-Gln-Gly-NH.sub.2, Lys-Gln-Gly-NH.sub.2, Ala-Leu-Gly-NH.sub.2,
Gly-Val-Gly-NH.sub.2, Val-Gly-Gly-NH.sub.2, Ala-Ser-Gly-NH.sub.2,
Ser-Leu-Gly-NH.sub.2, and Ser-Pro-Thr-NH.sub.2. Additionally, a
method of making a pharmaceutical is provided in which the peptide
agent identified by the methods above are incorporated in a
pharmaceutical.
[0025] Another approach to make a pharmaceutical is also provided,
which involves administering to a cell an effective amount of a
peptide in amide form having the formula
X.sub.1X.sub.2X.sub.3-NH.sub.2, wherein X.sub.1, X.sub.2, and
X.sub.3 are any amino acid and said peptide is not
Gly-Pro-Gly-NH.sub.2, detecting an inhibition of viral replication
in the cell, and incorporating the peptide that causes inhibition
of viral replication into the pharmaceutical. This method can
involve the use of a peptide that is selected from the group
consisting of peptides having the formula Gly-Lys-Gly-NH.sub.2,
Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2, Lys-Gln-Gly-NH.sub.2,
Ala-Leu-Gly-NH.sub.2, Gly-Val-Gly-NH.sub.2, Val-Gly-Gly-NH.sub.2,
Ala-Ser-Gly-NH.sub.2, Ser-Leu-Gly-NH.sub.2, and
Ser-Pro-Thr-NH.sub.2. Further, the method above can involve the
step of incorporating an antiviral compound selected from the group
consisting of nucleoside analogue reverse transcriptase inhibitors,
nucleotide analogue reverse transcriptase inhibitors,
non-nucleoside reverse transcriptase inhibitors, and protease
inhibitors into the pharmaceutical. Additionally, the method above
can involve the step of incorporating a carrier into the
pharmaceutical.
[0026] In another embodiment, a composition for inhibiting viral
replication in host cells infected with a virus includes an
effective amount of a peptide having the formula
X.sub.1X.sub.2X.sub.3-R, wherein X.sub.1, X.sub.2, and X.sub.3 are
any amino acid and said peptide is not Gly-Pro-Gly-NH.sub.2,
wherein R is a modulation group attached to the carboxy-terminus of
said peptide and R comprises an amide group or other moiety having
similar charge and steric bulk and wherein said composition
inhibits viral replication by interrupting viral capsid assembly.
This composition can be a peptide selected from the group
consisting of peptides having the formula Gly-Lys-Gly-NH.sub.2,
Arg-Gln-Gly-NH.sub.2, Cys-Gln-Gly-NH.sub.2, Lys-Gln-Gly-NH.sub.2,
Ala-Leu-Gly-NH.sub.2, Gly-Val-Gly-NH.sub.2, Val-Gly-Gly-NH.sub.2,
Ala-Ser-Gly-NH.sub.2, Ser-Leu-Gly-NH.sub.2, and
Ser-Pro-Thr-NH.sub.2. Desirably, X.sub.3 is glycine in these
embodiments.
[0027] Additionally, the composition above can include a peptide
that has the formula
X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.1X.su-
b.2X.sub.3-R, wherein X.sub.4, X.sub.5, X.sub.6, X.sub.7, X.sub.8,
X.sub.9, and X.sub.10 are any amino acid and wherein any one, two,
three, four, five, six, or seven amino acids is absent, wherein R
is a modulation group attached to the carboxy-terminus of said
peptide and R comprises an amide group or other moiety having
similar charge and steric bulk. Preferably, the composition above
includes a peptide X.sub.1X.sub.2X.sub.3 that is found in the amino
acid sequence of the capsid protein of the virus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a graph representing the results from an HIV
infectivity study conducted in HUT78 cells.
[0029] FIG. 2 is a composite of electron micrographs of untreated
HIV particles.
[0030] FIG. 3 is a composite of electron micrographs of HIV
particles that have been contacted with the protease inhibitor
Ritonavir.
[0031] FIG. 4 is a composite of electron micrographs of HIV
particles that have been contacted with GPG-NH.sub.2.
[0032] FIG. 5 illustrates an alignment of the protein sequence
corresponding to the carboxyl terminus of the HIV-1 p24 protein
(residues 146-231) and protein sequences of HIV-2, SIV, Rous
Sarcoma virus (RSV), human T cell leukemia virus-type 1 (HTLV-1),
mouse mammary tumor virus (MMTV), Mason-Pfizer monkey virus (MPMV),
and Moloney murine leukemia virus (MMLV). The bar represents the
major homology region(MHR).
[0033] FIG. 6 is a graph of p24 (pg/ml) detected in the supematent
of HIV infected cells that were cultured in the presence and
absence of GPG-NH2, Ritonavir (Rito), AZT, or combinations of these
agents.
[0034] FIG. 7 illustrates a thin layer chromatography support
having separated thereon non-protein bound radioactive labeled
compounds from rat urine and rat plasma (rp) sampled after oral
feeding of .sup.14C-GPG-NH.sub.2, as well as human plasma (Hp)
incubated with .sup.14C-GPG-NH.sub.2; the sampling times are
suffixed and R1-R13 serve to indicate the position of identified
radioactive compounds.
[0035] FIG. 8 is a thin layer chromatography support having
separated thereon .sup.14C-GPG-NH.sub.2 that was treated with 0.1N
HCl and 50 mM KCl and sampled over a 24 hour period; the numbers
represent the time of acid exposure and the R serves to indicate
the position of the identified .sup.14C-GPG-NH.sub.2.
[0036] FIG. 9 is a graph of the partition of .sup.14C-GPG-NH.sub.2
and its metabolites in rat blood.
[0037] FIG. 10 is a graph of the elimination of radioactivity from
the plasma fraction of rats that were orally fed
.sup.14C-GPG-NH.sub.2.
[0038] FIG. 11 shows crude and fractionated rat plasma proteins
that were separated on a 10% SDS/PAGE; 8-f3 refers to the third
fraction taken from a sample at 8 hours after administration of
.sup.14C-GPG-NH.sub.2, 8-f1 refers to the first fraction taken from
a sample at 8 hours after administration of .sup.14C-GPG-NH.sub.2,
4-f3 refers to the third fraction taken from a sample at 4 hours
after administration of .sup.14C-GPG-NH.sub.2, 4-f1 refers to the
first fraction taken from a sample at 4 hours after administration
of 14C-GPG-NH.sub.2, 8 h, 4 h, 2 h, and 1 h refer to the time the
sample was taken after administration of .sup.14C-GPG-NH.sub.2, and
B1-B4 serve to indicate the position of identified proteins.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0039] The inventor has discovered that modified small peptides
having sequences that correspond to viral capsid proteins prevent
and/or inhibit viral infection by interrupting proper nucleocapsid
formation. Such peptides are usefil in the treatment of viral
disease, particularly in HIV/AIDS afflicted subjects, and as
preventive agents for patients at-risk of viral infection,
particularly HIV infection, and for use with medical devices where
the risk of exposure to virus is significant.
[0040] In the disclosure below, the inventor demonstrates that
small peptides in amide form that have a sequence that corresponds
to viral proteins, such as GPG-NH.sub.2, GKG-NH.sub.2,
CQG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2, ALG-NH.sub.2,
GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2, SLG-NH.sub.2, and
SPT-NH.sub.2 inhibit the replication of viruses, such as HIV-1,
HIV-2, and SIV, as measured by viral infectivity assays that
monitor the amount of capsid protein or reverse transcriptase
activity present in culture supernatent. Further, the inventor
presents evidence that these small peptides inhibit viral
infectivity by a V3 loop independent mechanism at a stage
subsequent to DNA, RNA, and protein synthesis.
[0041] Electron microscopic images of HIV particles treated with
small peptides reveal that this novel class of antiviral agent
interrupts proper capsid assembly in a manner distinct from
protease inhibitors. Further, in vitro binding assays reveal that
the small peptides bind to the major capsid protein (p24) of HIV-1.
Because the sequences of several viral capsid proteins are known,
such as members of arenavirus, rotavirus, orbivirus, retrovirus,
papillomavirus, adenovirus, herpesvirus, paramyxovirus, myxovirus,
and hepadnavirus families, several small peptides that correspond
to these sequences can be selected and rapidly screened to identify
which ones effectively inhibit and/or prevent viral infection by
using the viral infectivity assays or electron microscopy
techniques or both described herein, or modifications of these
assays as would be apparent to those of skill in the art given the
present disclosure.
[0042] Several approaches to make biotechnological tools and
pharmaceutical compositions comprising small peptides and
peptidomimetics (collectively referred to as "peptide agents") that
correspond to sequences of viral capsid proteins are given below.
Although preferable peptide agents are tripeptides having an amide
group at their carboxy termini, such as GPG-NH.sub.2, GKG-NH.sub.2,
CQG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2, ALG-NH.sub.2,
GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2, SLG-NH.sub.2, and
SPT-NH.sub.2, the inventor also provides compositions and methods
of inhibiting viral replication in host cells, including HIV
replication in host cells, comprising a peptide in amide form
having the formula X.sub.1, X.sub.2, X.sub.3-NH.sub.2 or the
formula X4, X.sub.5, X.sub.1, X.sub.2, X.sub.3-NH.sub.2, wherein
X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5 are any amino acid
and wherein any one or two amino acids can be absent. Preferred
embodiments have a glycine residue as X.sub.3. In some embodiments,
the peptide agents are provided in monomeric form; in others, the
peptide agents are provided in multimeric form or in multimerized
form. Support-bound peptide agents are also used in several
embodiments. Pharmaceutical compositions comprising peptide agents
are administered as therapeutics or prophylactics or both for the
treatment and/or prevention of viral disease, preferably, HIV
infection. In some embodiments, the pharmaceutical compositions
comprising peptide agents are administered in combination with
other antiviral treatments including nucleoside analogue reverse
transcriptase inhibitors, nucleotide analogue reverse transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors, and
protease inhibitors. The inventor also provides evidence that small
peptides are resistant to acid hydrolysis, that a significant
amount of small peptide is effectively delivered to blood, plasma,
and organ tissue when administered to test subjects, and that the
administration of large doses of small peptides to test subjects is
relatively nontoxic.
[0043] Additionally, the inventor discloses several methods of
identifying a peptide agent that inhibits or prevents viral
replication or interrupts viral capsid assembly or both. By one
approach, an effective amount of a peptide agent is contacted with
cells infected with a virus and the cells are analyzed for viral
replication or the presence of viral products. Further, by using
electron microscopy, the ability of a peptide agent to interupt
capsid assembly can be readily determined. Still further, methods
are disclosed that identify a peptide agent that binds to a capsid
protein (e.g., p24) and thereby interupts capsid assembly and,
thus, viral replication. Accordingly, a capsid protein (e.g., p24)
is contacted with a peptide agent, for example a peptide in amide
form having the formula X.sub.1, X.sub.2, X.sub.3, wherein X.sub.1,
X.sub.2, and X.sub.3 are any amino acid, and a complex comprising
the capsid protein (e.g., p24) bound with the peptide agent is
identified. Reaction mixtures having a viral protein (e.g., p24)
and a peptide agent and a biomolecular complex having a viral
protein (e.g., p24) joined to a peptide agent are also taught in
the present disclosure.
[0044] The amide form of small peptides listed in Table 1 below
were tested. Many of these small peptides were selected and
synthesized because they either fully or partially correspond to
sequences in HIV and/or SIV viral proteins. The small peptides of
Table 1 were synthesized according to the method disclosed in
Example 1 below, but could of course be synthesized by any method
known in the art.
1TABLE 1 Amino Acid Sequence of Peptides Tested Leu-Lys-Ala (LKA)
Arg-Gln-Gly (RQG) Iso-Leu-Lys (ILK) Lys-Gln-Gly (KQG) Gly-Pro-Gln
(GPQ) Ala-Leu-Gly (ALG) Gly-His-Lys (GHK) Gly-Val-Gly (GVG)
Gly-Lys-Gly (GKG) Val-Gly-Gly (VGG) Ala-Cys-Gln (ACQ) Ala-Ser-Gly
(ASG) Cys-Gln-Gly (CQG) Ser-Leu-Gly (SLG) Ala-Arg-Val (ARV)
Ser-Pro-Thr (SPT) Lys-Ala-Arg (KAR) Gly-Ala-Thr (GAT) His-Lys-Ala
(HKA) Lys-Ala-Leu (KAL) Gly-Pro-Gly (GPG) Abbreviations Used:
Leu--Leucine Lys--Lysine Gln--Glutamine Ala--Alanine His--Histidine
Ileu--Isoleucine Cys--Cysteine Gly--Glycine Pro--Proline
Arg--Arginine Val--Valine Thr--Threonine Ser--Serine
EXAMPLE 1
[0045] In this example, the approaches used to obtain the small
peptides listed above are disclosed. Several tripeptides were
chemically synthesized with an automated peptide synthesizer (Syro,
Multisyntech, Tubingen, Germany). The synthesis was run using
9-fluorenylmethoxycarbony- l (fmoc) protected amino acids
(Milligen, Bedford, Mass.) according to standard protocols. All
peptides were lyophilized and then disolved at the appropriate
concentration in phosphate-buffered saline (PBS). The peptides were
analyzed by reverse phase high performance liquid chromatography
(RP-HPLC) using a PepS-15 C18 column (Pharmacia, Uppsala,
Sweden).
[0046] In many embodiments, peptides having a modulation group
attached to the carboxy-terminus of the peptide ("modified
peptides") were used. In some cases, the modified peptides were
created by substituting an amino group for the hydroxyl residue
normally present at the terminal carboxyl group of a peptide. That
is, instead of a terminal COOH, the peptides were synthesized to
have CO--NH.sub.2. For example, preferred small peptides include
glycyl-lysyl-glycine amide (GKG-NH.sub.2),
cystyl-glutaminyl-glycine amide (CQG-NH.sub.2),
glycyl-prolyl-glycine amide (GPG-NH.sub.2),
arginyl-glutaminyl-glycine amide (RQG-NH.sub.2),
lysyl-glutaminyl-glycine amide (KQG-NH.sub.2),
alanyl-leucyl-glycine amide (ALG-NH.sub.2), glycyl-valyl-glycine
amide (GVG-NH.sub.2), valyl-glycyl-glycine amide (VGG-NH.sub.2),
alanyl-seryl-glycine amide (ASG-NH.sub.2), seryl-leucyl-glycine
amide (SLG-NH.sub.2), and seryl-prolyl-threonine amide
(SPT-NH.sub.2). In addition to those synthesized, many tripeptides
were also purchased from Bachem AG, Switzerland, including but not
limited to, GKG-NH.sub.2, CQG-NH.sub.2, and GPG-NH.sub.2.
[0047] In the toxicology experiments and experiments that evaluated
the effects of small peptide treatment in combination with
conventional antiviral therapies, the peptides were obtained as
follows. For the initial experiments, solid phase peptide synthesis
was performed using an Applied Biosystems 430A peptide synthesizer
(Applied Biosystems, Foster City, Calif.). Each synthesis used a
p-methylbenzylhydrylamine solid phase support resin (Peptide
International, Louisville, Ky.) yielding a carboxyl terminal amide
when the peptides are cleaved off from the solid support by acid
hydrolysis. All amino acids for use in synthesis contained
t-butylcarbonyl groups protecting the .alpha.-NH2 group and were
obtained from Novabiochem AG, Switzerland. The protecting groups
were removed from the synthesized peptides that were cleaved from
the solid support resin by treatment with trifluoromethane sulfonic
acid, giving peptides with an amino (--NH2) modulation group
instead of a hydroxyl (--OH) group at the carboxyl terminus. Prior
to use, the peptides were purified by reverse phase high
performance liquid chromatography and sequenced on an Applied
Biosystems 473A peptide sequencer. In addition, the tripeptide GPG
having either an amide (CO--NH.sub.2; GPG-NH2) or carboxyl (COOH;
GPG-OH) terminus was purchased from Bachem AG, Switzerland.
[0048] In the disclosure below, several assays that were used to
identify small peptides that inhibit HIV-1, HIV-2, and SIV
infection are described.
[0049] HIV and SIV infectivity assays
[0050] The peptides made according to Example 1 were used in
several HIV-1, HIV-2, and SIV infection assays. The efficiency of
HIV-1, HIV-2, and SIV infection was monitored by reverse
transcriptase activity, the concentration of p24 protein in the
cell supernatent, and by microscopic evaluation of HIV-1 syncytia
formation.
[0051] In initial experiments, several tripeptides were screened
for the ability to inhibit HIV-1, HIV-2, and SIV infection in H9
cells. Once inhibitory tripeptides were identified, more specific
assays were conducted to determine the effect of varying
concentrations of the selected tripeptides and combination
treatments (e.g., the use of more than one tripeptide in
combination).
[0052] In the example below, an approach that was used to screen
several tripeptides for their ability to inhibit HIV-1, HIV-2, and
SIV infection is disclosed.
EXAMPLE 2
[0053] In this example, the methods that were used to analyze the
ability of various tripeptides to inhibit HIV-1, HIV-2, and SIV
replication are disclosed. In Experiments 1 and 2, approximately
200,000 H9 cells were infected with HIV-1, HIV-2 or SIV at 25
TCID.sub.50 to test the inhibitory effect of the following
synthesized tripeptides LKA-NH.sub.2, ILK-NH.sub.2, GPQ-NH.sub.2,
GHK-NH.sub.2, GKG-NH.sub.2, ACQ-NH.sub.2, CQG-NH.sub.2,
ARV-NH.sub.2, KAR-NH.sub.2, HKA-NH.sub.2, GAT-NH.sub.2,
KAL-NH.sub.2, and GPG-NH.sub.2. Accordingly, the H9 cells were
resuspended with or without the different peptides (approximately
100 .mu.M) in 1 ml of RPMI 1640 medium supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (FBS), penicillin (100 u/ml),
and streptomycin (100 u/ml), all available through GIBCO, and
Polybrene (2 .mu.g/ml), available through Sigma. Thereafter,
viruses were added at 25 TCID.sub.50 in a volume of 20-30.mu.l.
Cells were incubated with virus at 37.degree. C. for 1 hr then
pelleted at 170.times.g for 7 minutes. The cells were then washed
three times in RPMI medium without peptides at room temperature and
pelleted at 170.times.g for 7 minutes, as above. After the final
wash, the cells were resuspended in RPMI culture medium containing
the peptides in a 24-well plate (Costar corporation) and were kept
at 37.degree. C. in 5% CO.sub.2 with humidity.
[0054] Culture supernatants were collected and analyzed when the
medium was changed at 4, 7, 10, and 14 days post infection. To
monitor the replication of virus, reverse transcriptase (RT)
activity in the supernatants was assayed using a commercially
available Lenti-RT activity kit. (Cavidi Tech, Uppsala, Sweden).
The amount of RT was determined with the aid of a regression line
of standards. The results are presented as absorbance values (OD)
and higher absorbance indicates a higher protein concentration and
greater viral infection. Syncytium formation was also monitored by
microscopic examination. Tables 2 and 3 show the absorbance values
of the cell culture supernatants of Experiments 1 and 2
respectively.
[0055] In Experiment 3, (Table 4), approximately 200,000 H9 cells
were infected with HIV-1, HIV-2 or SIV at 25 TCID.sub.50 to test
the inhibitory effect of different concentrations of peptides
GPG-NH.sub.2, GKG-NH.sub.2 and CQG-NH.sub.2 and combinations of
these peptides (the indicated concentration corresponds to the
concentration of each tripeptide). As above, H9 cells were
resuspended with or without the different peptides at varying
concentrations in 1 ml of RPMI 1640 medium supplemented with 10%
(v/v) heat-inactivated fetal bovine serum (FBS), penicillin (100
u/ml), and streptomycin (100 u/ml), and Polybrene (2 .mu.g/ml).
Thereafter, viruses were added at 25 TCID.sub.50 in a volume of
20-30 .mu.l. Cells were incubated with the indicated virus at
37.degree. C. for 1 hr then pelleted at 170.times.g for 7 minutes.
The cells were then washed three times in RPMI medium without
peptides at room temperature and pelleted at 170.times.g for 7
minutes, as above. After the final wash, the cells were resuspended
in RPMI culture medium containing the peptides in a 24-well plate
(Costar corporation) and kept at 37.degree. C. in 5% CO.sub.2 with
humidity.
[0056] Culture supernatants were collected when the medium was
changed at 4, 7, and 11 days post infection. As above, the
replication of each virus was monitored by detecting reverse
transcriptase (RT) activity in the supernatants using the Lenti-RT
activity kit. (Cavidi Tech). The amount of RT was determined with
the aid of a regression line of standards. The results are
presented as absorbance values (OD) and higher absorbance indicates
a higher protein concentration and greater viral infection. Table 4
shows the absorbance values of the cell culture supernatents of
Experiment 3.
[0057] In Experiment 4, (Table 5) approximately 200,000 H9 cells
were infected with HIV-1 at 25 TCID.sub.50 to test the inhibitory
effect of different concentrations of peptides GPG-NH.sub.2,
GKG-NH.sub.2 and CQG-NH.sub.2 and combinations of these peptides
(the indicated concentration corresponds to the total concentration
of tripeptide). As above, H9 cells were resuspended with or without
the different peptides at varying concentrations in 1 ml of RPMI
1640 medium supplemented with 10% (v/v) heat-inactivated fetal
bovine serum (FBS), penicillin (100 u/ml), and streptomycin (100
u/ml), and Polybrene (2 .mu.g/ml). Thereafter, viruses were added
at 25 TCID.sub.50 in a volume of 20-30.mu.l. Cells were incubated
with the indicated virus at 37.degree. C. for 1 hr then pelleted at
170.times.g for 7 minutes. The cells were then washed three times
in RPMI medium without peptides at room temperature and pelleted at
170.times.g for 7 minutes, as above. After the final wash, the
cells were resuspended in RPMI culture medium containing the
peptides in a 24-well plate (Costar corporation) and kept at
37.degree. C. in 5% CO.sub.2 with humidity.
[0058] Culture supernatants were collected when the medium was
changed at 4, 7, and 11 days post infection. As above, the
replication of each virus was monitored by detecting reverse
transcriptase (RT) activity in the supernatants using the Lenti-RT
activity kit. (Cavidi Tech). The amount of RT was determined with
the aid of a regression line of standards. The results are
presented as absorbance values (OD) and higher absorbance indicates
a higher protein concentration and greater viral infection. Table 5
shows the absorbance values of the cell culture supernatents of
Experiment 4. The supernatant analyzed at day 11 was diluted 5-fold
so that detection could be more accurately determined.
[0059] In Experiment 5, (Table 6) approximately 200,000 H9 cells
were infected with HIV-1 at 25 TCID.sub.50 to test the inhibitory
effect of different concentrations of peptides GPG-NH.sub.2,
GKG-NH.sub.2 and CQG-NH.sub.2 and combinations of these peptides.
As above, H9 cells were resuspended with or without the different
peptides at varying concentrations in 1 ml of RPMI 1640 medium
supplemented with 10% (v/v) heat-inactivated fetal bovine serum
(FBS), penicillin (100 u/ml), streptomycin (100 u/ml), and
Polybrene (2 .mu.g/ml). Thereafter, viruses were added at 25
TCID.sub.50 in a volume of 20-30 .mu.l. Cells were incubated with
the indicated virus at 37.degree. C. for 1 hr then pelleted at
170.times.g for 7 minutes. The cells were then washed three times
in RPMI medium without peptides at room temperature and pelleted at
170.times.g for 7 minutes, as above. After the final wash, the
cells were resuspended in RPMI culture medium containing the
peptides in a 24-well plate (Costar corporation) and kept at
37.degree. C. in 5% CO.sub.2 with humidity.
[0060] Culture supernatants were collected when the medium was
changed at 4, 7, and 14 days post infection. The replication of
each virus was monitored by detecting the presence of p24 in the
supernatants. HIV p24 antigen was determined using a commercially
available HIV p24 antigen detection kit (Abbott). The results are
presented as absorbance values (OD) and higher absorbance indicates
a higher protein concentration and greater viral infection. In some
cases, serial dilutions of the supernatants were made so as to more
accurately detect p24 concentration. Table 6 shows the absorbance
values of the cell culture supernatants of Experiment 5. As
discussed in greater detail below, it was discovered that the
tripeptides GPG-NH.sub.2, GKG-NH.sub.2 and CQG-NH.sub.2 and
combinations of these peptides effectively inhibit HIV-1, HIV-2,
and SIV infection.
[0061] In experiment 6 (Table 7 and FIG. 1), approximately 200,000
HUT78 cells were infected with HIV-1 at 25 TCID.sub.50 to test the
inhibitory effect of GPG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2,
ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2,
SLG-NH.sub.2, and SPT-NH.sub.2. The HUT cells were resuspended in
lml of RPMI 1640 medium supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (FBS, GIBCO), penicillin (100
u/ml), streptomycin (100 u/ml) and Polybrene (Sigma, 2 .mu.g/ml)
with or without the presence of the different small peptides (100
.mu.M) mentioned above. Thereafter, the HIV-1 virus was added at 25
TCID.sub.50 in a volume of 20 .mu.l. Cells were incubated with the
virus at 37.degree. C. for one hour and, subsequently, the cells
were pelleted at 170.times.g for seven minutes. The cells were then
washed three times in RPMI medium without peptides at room
temperature by cell sedimentation at 170.times.g for seven minutes,
as above. After the final wash, the cells were resuspended in RPMI
culture medium containing the peptides in 24-well plate (Costar
corporation) and were kept at 37.degree. C. in 5% CO.sub.2 with
humidity. Culture supernatants were collected when medium was
changed at day 4, 7, and 11 post infection and viral p24 production
was monitored by using an HIV-1 p24 ELISA kit (Abbott Laboratories,
North Chicago, USA). As discussed below, it was discovered that the
small peptides RQG-NH.sub.2, KQG-NH.sub.2, ALG-NH.sub.2,
GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2, SLG-NH.sub.2, and
SPT-NH.sub.2 effectively inhibit HIV-1 infection.
2TABLE 2 Experiment 1 - (peptides made on site) Day 10 RT
Tripeptide Day 7 RT HIV-1 (100 .mu.M) HIV-1 HIV-2 SIV HIV-1 HIV-2
SIV Syncytia LKA-NH.sub.2 0.568* 3.649 3.577 2.429 2.769 2.452 pos
ILK-NH.sub.2 0.365 3.467 3.180 2.033 2.791 2.255 pos GPQ-NH.sub.2
0.204 3.692 1.542 1.965 2.734 2.176 pos GHK-NH.sub.2 0.289 3.522
0.097 2.151 2.931 2.384 pos GKG-NH.sub.2 0.080 0.160 0.421 0.074
0.147 0.099 neg ACQ-NH.sub.2 0.117 3.418 1.241 0.904 2.753 2.746
pos CQG-NH.sub.2 0.091 0.217 0.747 0.108 0.296 0.110 neg
ARV-NH.sub.2 0.156 3.380 0.210 1.528 3.003 1.172 pos KAR-NH.sub.2
0.380 3.419 0.266 2.779 2.640 1.722 pos HKA-NH.sub.2 0.312 3.408
0.416 2.546 2.669 2.520 pos GAT-NH.sub.2 0.116 3.461 0.892 1.565
2.835 2.343 pos KAL-NH.sub.2 0.246 3.372 1.091 1.995 2.749 2.524
pos GPG-NH.sub.2 0.068 0.735 0.138 0.074 0.145 0.103 neg NO PEPTIDE
0.251 1.675 1.227 2.217 2.657 3.030 pos CONTROL *Values represent
opitcal density (OD)
[0062]
3TABLE 3 Experiment 2 - (peptides made on site) Day 10 RT
Tripeptide Day 7 RT HIV-1 (100 .mu.M) HIV-1 HIV-2 SIV HIV-1 HIV-2
SIV Syncytia LKA-NH.sub.2 0.894* 1.689 0.724 2.989 2.637 2.797 pos
ILK-NH.sub.2 0.581 1.692 0.515 2.950 2.557 2.632 pos GPQ-NH.sub.2
0.884 1.511 0.574 2.848 2.382 2.319 pos GHK-NH.sub.2 0.829 1.936
0.396 3.013 2.418 2.394 pos GKG-NH.sub.2 0.145 0.283 0.116 0.345
1.637 0.204 neg ACQ-NH.sub.2 0.606 1.661 0.612 2.831 2.505 2.606
pos CQG-NH.sub.2 0.143 1.241 0.120 1.546 2.501 1.761 neg
ARV-NH.sub.2 0.618 2.237 0.212 2.829 2.628 3.004 pos KAR-NH.sub.2
0.753 1.904 1.034 2.928 2.742 2.672 pos HKA-NH.sub.2 1.081 1.678
0.455 2.794 2.560 2.623 pos GAT-NH.sub.2 0.776 1.707 0.572 2.800
2.565 2.776 pos KAL-NH.sub.2 0.999 1.757 0.511 2.791 2.383 2.663
pos GPG-NH.sub.2 0.090 0.093 0.067 0.143 0.575 0.139 neg NO PEPTIDE
0.809 1.774 0.578 2.711 2.528 2.911 pos CONTROL *Values represent
opitcal density (OD)
[0063]
4TABLE 4 Experiment 3 - (peptides obtained from Bachem) Day 7 RT
Day 10 RT Tripeptide HIV-1 HIV-2 SIV HIV-1 HIV-2 SIV NO PEPTIDE
1.558* 1.718 1.527 2.521 2.716 2.091 CONTROL GPG-NH.sub.2 1.527
1.735 0.753 2.398 2.329 2.201 5 .mu.M GPG-NH.sub.2 0.239 0.252
0.197 0.692 1.305 0.779 20 .mu.M GKG-NH.sub.2 1.587 1.769 0.271
1.683 2.510 1.709 5 .mu.M GKG-NH.sub.2 1.616 1.759 1.531 2.036
2.646 2.482 20 .mu.M GKG-NH.sub.2 0.823 0.828 1.005 1.520 1.947
1.382 100 .mu.M CQG-NH.sub.2 1.547 1.760 1.159 2.028 2.466 2.821 5
.mu.M CQG-NH.sub.2 1.578 1.748 0.615 1.484 2.721 2.158 20 .mu.M
CQG-NH.sub.2 1.520 1.715 0.795 2.014 2.815 2.286 100 .mu.M
GPG-NH.sub.2 + GKG-NH.sub.2 1.430 1.738 1.131 1.998 2.770 2.131 5
.mu.M GPG-NH.sub.2 + GKG-NH.sub.2 0.129 0.244 0.123 0.164 1.110
0.309 20 .mu.M GPG-NH.sub.2 + CQG-NH.sub.2 1.605 1.749 1.737 1.866
2.814 2.206 5 .mu.M GPG-NH.sub.2 + CQG-NH.sub.2 0.212 0.194 0.523
0.397 1.172 0.910 20 .mu.M GKG-NH.sub.2 + CQG-NH.sub.2 1.684 1.717
1.725 1.848 2.778 2.949 5 .mu.M GKG-NH.sub.2 + CQG-NH.sub.2 1.490
1.792 1.670 1.891 2.799 2.889 20 .mu.M GPG-NH.sub.2 + GKG-NH.sub.2
1.652 1.743 1.628 1.999 2.777 2.659 5 .mu.M GPG-NH.sub.2 +
GKG-NH.sub.2 0.165 0.119 0.317 0.307 0.447 0.389 20 .mu.M *Values
represent opitcal density (OD)
[0064]
5TABLE 5 Experiment 4 - (peptides obtained from Bachem) Day 10 RT
Day 7 RT HIV-1 Tripeptide HIV-1 (1:5) NO PEPTIDE CONTROL 3.288*
1.681 GPG 5 .mu.M 2.970 1.107 GPG 15 .mu.M 0.894 0.095 GPG 45 .mu.M
0.177 0.034 GPG 100 .mu.M 0.150 0.033 GKG 5 .mu.M 3.303 1.287 GKG
15 .mu.M 3.551 1.530 GKG 45 .mu.M 3.126 0.410 CQG 5 .mu.M 2.991
1.459 CQG 15 .mu.M 2.726 1.413 CQG 45 .mu.M 3.124 1.364
GPG--NH.sub.2 + GKG--NH.sub.2 2.266 0.438 5 .mu.M GPG--NH.sub.2 +
GKG--NH.sub.2 0.216 0.044 15 .mu.M GPG--NH.sub.2 + CQG--NH.sub.2
2.793 0.752 5 .mu.M GPG--NH.sub.2 + CQG--NH.sub.2 0.934 0.110 15
.mu.M GkG--NH.sub.2 + CQG--NH.sub.2 3.534 1.305 5 .mu.M
GKG--NH.sub.2 + CQG--NH.sub.2 3.355 2.013 15 .mu.M GPG--NH.sub.2 +
GKG--NH.sub.2 + CQG--NH.sub.2 2.005 0.545 5 .mu.M GPG--NH.sub.2 +
GKG--NH.sub.2 + CQG--NH.sub.2 0.851 0.110 15 .mu.M *Values
represent optical density (OD)
[0065]
6TABLE 6 Experiment 5 - (peptides made on site) Tripeptide (.mu.M)
p24 (OD) p24 (pg/ml) reduction (%) Day 7 HIV-1 NO PEPTIDE CONTROL
1.093 .times. 10.sup.2 3.94 .times. 10.sup.4 0 GPG--NH.sub.2 (20)
1.159 4.21 .times. 10.sup.2 99 GPG--NH.sub.2 (100) 0.508 1.60
.times. 10.sup.2 100 GPG--NH.sub.2 (300) 0.557 1.80 .times.
10.sup.2 100 GKG--NH.sub.2 (100) 0.566 .times. 10.sup.1 1.83
.times. 10.sup.3 95 GKG--NH.sub.2 (300) 1.08 3.88 .times. 10.sup.2
99 GKG--NH.sub.2 (1000) 0.79 2.73 .times. 10.sup.2 100
CQG--NH.sub.2 (100) 1.51 .times. 10.sup.1 5.62 .times. 10.sup.3 86
CQG--NH.sub.2 (300) 0.59 .times. 10.sup.1 1.92 .times. 10.sup.3 95
CQG--NH.sub.2 (1000) 0.91 3.20 .times. 10.sup.2 99 combined* 0.65
2.17 .times. 10.sup.2 100 Day 14 HIV-1 NO PEPTIDE CONTROL 0.46
.times. 10.sup.4 1.41 .times. 10.sup.6 0 GPG--NH.sub.2 (20) 1.12
.times. 10.sup.2 4.06 .times. 10.sup.4 97 GPG--NH.sub.2 (100) 1.76
6.63 .times. 10.sup.2 100 GPG--NH.sub.2 (300) 1.35 4.98 .times.
10.sup.2 100 GKG--NH.sub.2 (100) 1.48 .times. 10.sup.3 5.51 .times.
10.sup.5 61 GKG--NH.sub.2 (300) 0.33 .times. 10.sup.1 8.70 .times.
10.sup.2 100 GKG--NH.sub.2 (1000) 0.11 .times. 10.sup.1 2.40
.times. 10.sup.2 100 CQG--NH.sub.2 (100) 0.48 .times. 10.sup.4 1.47
.times. 10.sup.6 0 CQG--NH.sub.2 (300) 0.11 .times. 10.sup.2 2.40
.times. 10.sup.3 100 CQG--NH.sub.2 (1000) 0.13 .times. 10.sup.1
2.80 .times. 10.sup.2 100 combined* 1.01 3.61 .times. 10.sup.2 100
*100 .mu.M GPG--NH.sub.2 + GKG--NH.sub.2 + CQG--NH.sub.2 *Values
represent opitcal density (OD)
[0066]
7TABLE 7 Experiment 6 - (peptides made on site) Tripeptide (100
.mu.M) Day 7 HIV-1 p24 (pg/ml) reduction (%) NO PEPTIDE CONTROL 2.0
.times. 10.sup.4 0 GPG--NH.sub.2 5.6 .times. 10.sup.2 97
RQG--NH.sub.2 1.13 .times. 10.sup.2 99 KQG--NH.sub.2 1.54 .times.
10.sup.2 99 ALG--NH.sub.2 0.42 .times. 10.sup.2 100 GVG--NH.sub.2
1.5 .times. 10.sup.4 25 VGG--NH.sub.2 1.0 .times. 10.sup.4 50
ASG--NH.sub.2 1.5 .times. 10.sup.4 25 SLG--NH.sub.2 1.14 .times.
10.sup.2 99 SPT--NH.sub.2 1.5 .times. 10.sup.4 25
[0067] Small peptides inhibit and/or prevent HIV-1, HIV-2, and SIV
infection
[0068] Of the small pepti des listed in Table 1, GPG-NH.sub.2,
GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2,
ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2,
SLG-NH.sub.2, and SPT-NH.sub.2 inhibited and/or prevented HIV-1
infection and GKG-NH.sub.2, CQG-NH.sub.2, and GPG-NH.sub.2 were
also shown to inhibit or prevent HIV-2 and SIV infection. It should
be understood that the small peptides RQG-NH.sub.2, KQG-NH.sub.2,
ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2,
SLG-NH.sub.2, and SPT-NH.sub.2 were not analyzed for their ability
to prevent or inhibit HIV-2 or SIV infection but, given the fact
that HIV-2 and SIV share significant homology in capsid protein
structure at the region to which the small peptides GPG-NH.sub.2,
GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2,
ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2,
SLG-NH.sub.2, and SPT-NH.sub.2 correspond, an inhibition or
prevention of HIV-2 or SIV infection or both is expected.
[0069] The results for Experiments 1-6 (shown in Tables 2-7 and
FIG. 1), demonstrate that small peptides in amide form that
correspond to viral capsid protein sequence having a glycine as the
carboxyterminal amino acid, GPG-NH.sub.2, GKG-NH.sub.2,
CQG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2, ALG-NH.sub.2,
GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2, and SLG-NH.sub.2,
inhibited or prevented HIV infection. Peptides containing a
carboxyterminal alanine residue, Leu-Lys-Ala (LKA) and His-Lys-Ala
(HKA) or a carboxyterminal glutamine residue, Gly-Pro-Gln (GPQ) and
Ala-Cys-Gln (ACQ) did not prevent HIV infection. Glycine at the
amino terminus was not an inhibitory factor, however, because the
peptides with an amino terminal glycine residue, Gly-Pro-Gln (GPQ),
Gly-His-Lys (GHK), and Gly-Ala-Thr (GAT) failed to prevent
infection and HIV-1 syncytia formation. Further, peptides with
other uncharged polar side chains such as Gly-Pro-Gln (GPQ),
Ala-Cys-Gln (ACQ), and Gly-Ala-Thr (GAT) or non-polar side chains
at the carboxy terminus such as Ala-Arg-Val (ARV), His-Lys-Ala
(HKA), and Lys-Ala-Leu (KAL), and Leu-Lys-Ala (LKA) failed to
prevent infection. Although a glycine residue at the carboxy
terminus appears to be associated with the inhibition of HIV and
SIV infection, other amino acid residues or modified amino acid
residues at the carboxy terminus of a small peptide can also
inhibit HIV and SIV infection. For example, it was shown that
Ser-Pro-Thr (SPT) inhibited or prevented HIV-1 infection.
[0070] In some experiments it appeared that the effect of the small
peptides on HIV-1, HIV-2, and SIV infection was concentration and
time dependent. Concentrations of GKG-NH.sub.2, CQG-NH.sub.2, and
GPG-NH.sub.2 and combinations thereof, as low as 5 .mu.M and 20
.mu.M were shown to be effective at reducing HIV-1, HIV-2, and SIV
infection. At 100 .mu.M or greater, however, the tripeptides
GKG-NH.sub.2, CQG-NH.sub.2, and GPG-NH.sub.2 and combinations
thereof more efficiently inhibited HIV-1, HIV-2, and SIV infection.
As shown in Table 6, 300 .mu.M of GKG-NH2 and CQG-NH2 reduced HIV-1
infectivity by almost 100%, as detected by the presence of p24
antigen in cell supematents. The percent reduction tabulated in
Table 6 was calculated by dividing amount of p24 antigen detected
in the peptide-treated sample by the amount of p24 antigen detected
in the control sample, multiplying this dividend by 100 to obtain a
percentage, and subtracting the dividend percentage by 100%. For
example, the percent reduction exhibited by GPG-NH.sub.2 is: 1 5.6
.times. 10 2 2.0 .times. 10 4 .times. 100 = 3 % and 100 % - 3 % =
97 % .
[0071] In the first five experiments (Tables 2-6) it was shown that
the tripeptides GKG-NH.sub.2, CQG-NH.sub.2, and GPG-NH.sub.2 and
combinations thereof, inhibit HIV-1, HIV-2, and SIV infection at
concentrations equal to or greater than 5 .mu.M.
[0072] In the sixth experiment (Table 7 and FIG. 1), it was shown
that the small peptides RQG-NH.sub.2, KQG-NH.sub.2, ALG-NH.sub.2,
GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2, SLG-NH.sub.2, and
SPT-NH.sub.2 effectively inhibit and/or prevent HIV-1 infection at
100 .mu.M. As shown in Table 7, a nearly 100% reduction of virus,
as measured by the amount of capsid protein p24 in the supernatent,
was achieved with the small peptides RQG-NH.sub.2, KQG-NH.sub.2,
ALG-NH.sub.2, and SLG-NH.sub.2. The percent reduction of p24 shown
in Table 7 was calculated as described for Table 6, above. Although
GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2, and SPT-NH.sub.2 were
less effective at inhibiting or preventing HIV-1 infection at 100
.mu.M, it is believed that the tripeptides are more effective at
higher concentrations. The data presented in experiments 1-6, shown
in Tables 2-7 and FIG. 1, demonstrate that small peptides that
correspond to sequences of a viral capsid protein are effective
antiviral agents over a wide-range of concentrations.
[0073] In order to better understand small peptide-mediated viral
inhibition, several studies on DNA synthesis, RNA synthesis, and
protein expression were conducted. These experiments are discussed
below.
[0074] Small peptides inhibit viral infectivity at a stage
subsequent to DNA synthesis, RNA synthesis, and protein
expression
[0075] To study proviral DNA and viral RNA synthesis, DNA and RNA
from HIV-1 infected H-9 cells cultured in the presence of a small
peptide were prepared at various time points (0-48 h). Southern
blot analysis revealed that HIV-1 DNA was synthesized in the
presence of GPG-NH.sub.2 and the amount of proviral DNA was almost
equal at various concentrations (0-2,000 .mu.M) of the small
peptide during the first 24 h. These results prove that small
peptides, such as GPG-NH.sub.2, have no inhibitory effect on HIV-1
entry and DNA synthesis, and coincide with the finding that the
small peptides do not inhibit HIV-1 reverse transcriptase
activity.
[0076] By Northern blot analysis, three RNA bands (9.2, 4.3, 2.0
kb) were detected 24-48 h after infection. The 9.2 kb RNA acts as
both the genomic RNA and as the mRNA for the gag and pol genes. The
4.3 kb singly-spliced RNA represents at least the env gene, and the
multiply-spliced 2 kb RNA encodes for the regulatory genes.
GPG-NH.sub.2 at 20 .mu.M had no inhibitory effect on expression of
these RNAs up to 48 h post infection. At 200 .mu.M and 2,000 .mu.M
a reduction of HIV-1 RNA was noticed 48 h after infection that
probably reflects inhibition of the second replication cycle. These
results established that small peptides do not inhibit HIV-1
replication at the transcription step, nor do they affect the
splicing of transcripts.
[0077] In the experiments designed to determine whether HIV-1
properly expresses protein in the presence of small peptides, no
significant effect on protein expression or modification was
observed. In one experiment, however, an aberrant migration of
gp160/gp120 on a polyacrylamide gel was seen. In the presence
GPG-NH.sub.2 (20 .mu.M or more), gp160 and/or gp120 was observed to
electrophorese to a position on a polyacrylamide gel representative
of a molecular weight of slightly less than 120,000 Da. This result
was not reproducible and a change in migration of the gag-proteins
p17 or p24 was not observed. Studies to analyze glycosylation of
the virus protein in the presence of GPG-NH.sub.2 showed that there
was no effect on either N- or O-linked glycosylation. Also,
glycosylation on recombinantly (in vaccinia virus) produced gp160
was not affected by GPG-NH.sub.2. Furthermore, GPG-NH.sub.2 was
found not to affect the activity of the HIV-1 specific
protease.
[0078] In the experiments presented above, it was demonstrated that
small peptides interfere with HIV infectivity at a late stage of
the HIV replicative cycle. GPG-NH.sub.2 was unable to disrupt DNA
synthesis, RNA synthesis, protein synthesis, and protein
glycosylation. In the following disclosure, more evidence that
small peptides, such as GPG-NH.sub.2, GKG-NH.sub.2, CQG-NH.sub.2,
RQG-NH.sub.2, KQG-NH.sub.2, ALG-NH.sub.2, GVG-NH.sub.2,
VGG-NH.sub.2, ASG-NH.sub.2, SLG-NH.sub.2, and SPT-NH.sub.2, inhibit
viral infection at a late stage in the replicative cycle is
disclosed and, in a broader sense, another technique that can be
used to screen other small peptides and derivatives thereof for the
ability to inhibit viral infection, such as HIV or SIV infection,
is provided. Accordingly, discussed below are several electron
microscopy experiments in which HIV-1 infected cells were incubated
in the presence and absence of a small peptide.
[0079] Small peptides interfere with assembly of the
nucleocapsid
[0080] Once it had been discovered that the small peptides
GPG-NH.sub.2, GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2,
KQG-NH.sub.2, ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2,
ASG-NH.sub.2, SLG-NH.sub.2, and SPT-NH.sub.2 inhibited HIV
infection, electron microscopy was used to further analyze HIV
infected cells that had been incubated with a small peptide. (See
FIGS. 2, 3, and 4). As shown in FIG. 4, electron microscopic
analysis revealed that contact with GPG-NH.sub.2 interrupted proper
viral nucleocapsid formation.
[0081] In this set of experiments, HUT78 cells were infected with
HIV-1 SF-2 virus at 300TCID.sub.50 for 1 hr at 37.degree. C.
Subsequently, the infected cells were washed and pelleted 3 times,
as described in Example 2. Thereafter, the cells were resuspended
in RPMI culture medium supplemented with 10% FBS, antibiotics (100
u/ml) and polybrene (3.2 .mu.g/ml). GPG-NH.sub.2 was then added
into the cell cultures 3, 5 or 7 days post infection at
concentration of 1 .mu.M or 10 .mu.M. A control sample was
administered 0.5.mu.M Ritonavir (a protease inhibitor).
[0082] The cells were cultured until day 14, at which point, the
cells were fixed in 2.5% glutaraldehyde by conventional means. The
fixed cells were then postfixed in 1% OSO4 and were dehydrated,
embedded with epoxy resins, and the blocks were allowed to
polymerize. Epon sections of virus infected cells were made
approximately 60-80 nm thin in order to accommodate the width of
the nucleocapsid. The sections were mounted to grids stained with
1.0% uranyl acetate and were analyzed in a Zeiss CEM 902 microscope
at an accelerating voltage of 80 kV. The microscope was equipped
with a spectrometer to improve image quality and a liquid nitrogen
cooling trap was used to reduce beam damage. The grids having
sections of control and GPG-NH.sub.2 incubated cells were examined
in several blind studies.
[0083] Electron microscopy of untreated HIV particles revealed the
characteristic conical-shaped nucleocapsid and enclosed uniformly
stained RNA that stretched the length of the nucleocapsid. (See
FIG. 2). In contrast, FIG. 3 presents two electron micrographs
showing several HIV-1 particles that were produced in the presence
of the viral protease inhibitor Ritonavir. Infected cells that had
been treated with Ritonavir exhibited malformed structures that did
not have a discemable nucleocapsid, as was expected. (See FIG. 3).
FIG. 4 presents electron micrographs showing viral particles that
had been produced in the presence of GPG-NH.sub.2. Cells having
HIV-1 particles that were treated with GPG-NH.sub.2 exhibited HIV-1
particles with discernable capsid structures that are distinct from
the Ritonavir-treated particles. More specifically, in some
tripeptide-treated viral particles, the conical-shaped capsid
structure appeared to be relatively intact but the RNA was amassed
in a ball-like configuration either outside the capsid or at the
top (wide-end) of the capsid. Still further, some capsids were
observed to have misshapen structures with little or no morphology
resembling a normal nucleocapsid and RNA was seen to be either
outside the structure or inside the structure at one end. From
these studies it was clear that small peptides interfered with
proper formation of the nucleocapsid and that this inhibition of
capsid development occurred at a step distinct from the action of
the protease inhibitor Ritonavir.
[0084] More evidence that tripeptides in amide form, such as
GPG-NH.sub.2, GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2,
KQG-NH.sub.2, ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2,
ASG-NH.sub.2, SLG-NH.sub.2, and SPT-NH.sub.2, interfere with capsid
assembly was revealed when a binding assay with p24 as a target
biomolecule was performed. The details of the p24 binding assay are
provided below.
[0085] Small peptides bind to the major capsid protein (p24)
[0086] Experiments were performed to directly study whether small
peptides have the ability to interact with the mature capsid
protein (CA) or p24 and thereby interfere with nucleocapsid
fonnation. In this set of experiments, a p24 binding assay was
performed that assessed the ability of radiolabeled GPG-NH.sub.2 to
bind to p24.
[0087] A dialysis-based binding assay was conducted using a
dialysis membrane with a pore size of less than 10 kD.
(Slide-A-Lyzer, Pierce). Fifty microliters of a 10 .mu.M stock of
the recombinant proteins p24 and gp120 (gifts from the AIDS
program, NCIB) and BSA (Sigma) were introduced into separate
dialysis membranes and the proteins were dialyzed at 4.degree. C.
for 2 days against a 500 ml solution composed of 150 mM NaCl and 50
mM Tris-HCl, pH 7.4 buffer and 27.5 .mu.M of .sup.14C-GPG-NH.sub.2
(Amersham Ltd. UK). Subsequently, ten or five microliter aliquots
of the dialyzed p24, gp120, and BSA were removed and mixed with 3
ml of ReadySafe (Beckman) in a scintillation vial. The C.sup.14 was
then detected by scintillation counting.
[0088] In Table 8, the results from a representative dialysis
experiment are provided. Notably, an association of p24 with
GPG-NH.sub.2 was observed upon dialysis equilibration. The amount
of radioactive GPG-NH.sub.2 associated with p24 was 7.5 times
greater than that present in the buffer. In contrast, no
appreciable amount of radioactive GPG-NH.sub.2, over the amount
present in the dialysis buffer, was associated with either gp120 or
BSA. These results prove that small peptides, such as GPG-NH.sub.2,
bind to p24 and through this interaction interupt proper
nucleocapsid formation.
8 TABLE 8 Sample: dialysis buffer p24 gp120 BSA .mu.Ci/ml 1.816
13.712 1.745 1.674 times buffer 1.000 7.551 0.961 0.922
[0089] In the following disclosure, additional evidence that small
peptides, such as GPG-NH.sub.2, GKG-NH.sub.2, CQG-NH.sub.2,
RQG-NH.sub.2, KQG-NH.sub.2, ALG-NH.sub.2, GVG-NH.sub.2,
VGG-NH.sub.2, ASG-NH.sub.2, SLG-NH.sub.2, and SPT-NH.sub.2, inhibit
HIV and SIV infection by a mechanism that is different from the way
that AZT or Ritonavir inhibit these viruses is provided.
[0090] Small peptides inhibit HIV-1 strains that are resistant to
AZT or Ritonavir
[0091] The ability of small peptides to inhibit HIV-1 strains that
are resistant to either the nucleoside analogue AZT or the protease
inhibitor Ritonavir. The HIV-1 resistant isolates were cultivated
in peripheral blood mononuclear cells (PBMC) and the supernatants
were collected as virus stocks. Titration of TCID.sub.50 (50%
tissue culture infectious dose) were performed on PBMC and the
titers were calculated according to the Reed and Muench formula.
400,000 PBMC were infected with 25 TCID.sub.50 of those viruses
(HIV-1 SF162 was used as a control) by adsorption in 37.degree. C.
for one hour then washed three times. The cells were resuspended in
culture medium containing either no drug, GPG-NH.sub.2 (100 .mu.M),
AZT (5 .mu.M), or Ritonavir (0.1 .mu.M) and incubated in 37.degree.
C., CO.sub.2 and humidity. Culture medium was changed every four
days and the HIV-1 p24 antigen protein in the supernatants was
monitored by ELISA (Table 9).
[0092] As shown in Table 9, GPG-NH.sub.2 had a potent anti-viral
effect on either AZT or Ritonavir resistant HIV strains. The
results in this experiment substantiate the data presented above
and prove that small peptides inhibit HIV replication at a
different stage than AZT and Ritonavir. Additionally, the treatment
of "street strains" of HIV was accomplished with GPG-NH.sub.2, as
disclosed in U.S. Pat. No. 5,627,035 to Vahlne et al., herein
incorporated by reference in its entirety. Given that they are also
derived from the sequence of the HIV viral protein, the peptides
GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2,
ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2,
SLG-NH.sub.2, and SPT-NH.sub.2 are also useful in the treatment of
AZT and Ritonavir resistant HIV infections, as well as street
strains of HIV.
9TABLE 9 Control GPG 100 .mu.M AZT 5 .mu.M Rito.sup.b 0.1 .mu.M
Types Number (%) (% reduction) (% reduction) (% reduction) low AZT
p7261 130000* <500 <500 nt (0) (100%) (100%) inter AZT p7163
107000 <500 22200 nt (0) (100%) (79%) high AZT p7227 114000
<500 68000 nt (0) (100%) (40%) low PI p7300 146000 <500
nt.sup.a 64000 (0) (100%) (56%) high PI p7141 114000 <500 nt
98000 (0) (100%) (14%) *HIV-1 p24 values are shown in pg/ml and are
averages of duplicate experiments taken 14 days post infection.
.sup.anot tested .sup.bRitonavir Low AZT: isolated with a low
resistance to AZT. Inter AZT: isolated with a intermediate
resistance to AZT. High AZT: isolated with a high resistance to
AZT. Low PI: isolated with a low resistance to protease inhibitor.
High PI: isolated with a high resistance to protease inhibitor.
[0093] In further support of the data presented above, several
HIV-1 infectivity experiments on an HIV-1 mutant that lacked the
GPG-motif in its V3 loop were performed. These experiments,
detailed below, established that GPG-NH.sub.2 -mediated HIV-1
inhibition occurs in a V3 loop independent fashion. The V3 loop of
HIV-1 env glycoprotein gp120 contains a conserved GPG sequence at
the tip of the loop that may be involved in virus replication. To
determine whether GPG-NH.sub.2 inhibited HIV-1 infection by
perturbing a V3 loop interaction, a V3 loop HIV-1 mutant provirus
was constructed. This mutant provirus lacking the GPG domain was
tested for its ability to infect cells in infectivity assays and
was analyzed by immunocytochemistry and electron microscopy. The
example below describes the construction of the mutant provirus,
the assay for HIV-1 infectivity, the determination of the structure
of the mutant viral particles by immunocytochemistry and electron
microscopy, and the discovery that GPG-NH.sub.2 inhibits the
infection by mutant viral particles.
EXAMPLE 3
[0094] A GPG-deleted provirus based on the infectious clone pBRu-2
was constructed using conventional techniques in molecular biology.
Escherichia coli DH5.alpha. and NM522 mutS were used for
sub-cloning, mutagenesis, and amplification of plasmid DNAs. The
puc18 plasmid was used as a vector for sub-cloning, and the HIV-1
proviral clone pBRu-2, which contains a full-length,
replication-competent clone of HIV-1/Bru (also designated as LAV,
LAI), was used to generate mutant virus.
[0095] The 2.7-kb SalI-to-BamHI fragment from pBRu-2, which encodes
the env gene, was sub-cloned into the SalI and BamHI sites of the
puc18 vector. GPG deletion was accomplished by site-directed
mutagenesis using the U.S.E. Mutagenesis Kit (Pharmacia). Two
oligonucleotides were required. The mutagenic oligonucleotide was
5'phosphorylated CGT ATC CAG AGG AGA GCA TTT GTT ACA ATA GG-3'
(obtained from Scandinavian Gene Synthesis AB, Stockholm, Sweden).
The selective oligonucleotide was 5'-phosphorylated GTG CCA CCT GTC
GAC TAA GAA ACC AT-3' and was designed to remove a unique
restriction site, AatII, in the puc18 vector. The wild-type DNA
was, thus, selectively eliminated from the mixed pool of wild-type
and mutant DNA by digestion with the corresponding endonuclease
AatII. The mutagenesis reaction products were transformed into E.
coli to amplify the plasmid DNAs. The mutation was verified by
Polymerase Chain Reaction (PCR) sequencing of the DNA using an
Automated Laser Fluorescent ALFTM DNA Sequencer (Pharmacia). The
SalI-BamHI fragment deleted from the oligonucleotide encoding GPG
was then cloned into pBRu-2 to generate the proviral plasmid. The
mutant DNAs from several bacterial colonies were amplified and
purified by QIAGEN Plasmid Kit. Two of them, mp8 and mp10 DNAs,
were transfected into cells.
[0096] The wild-type DNA and the mutant DNA, mp8 and mp10 were
transfected into Hela, HUT.sub.78, or MT-2 cells using DEAE-dextran
method. (Palker et al., Proc. Natl. Acad. Sci. USA 85: 1932-1936
(1988)). Briefly, 10.sup.6 cells were prepared 24 hours before
transfection. Then, the medium was removed and the cells were
washed once with pre-warmed phosphate-buffered- saline (PBS) and
once with TBS-D (Tris buffered saline-0.1% dextrose). Approximately
0.5 .mu.g DNA was mixed with TBS-D/DEAE-dextran and was added to
the cells followed by a 30 minute-incubation at 37.degree. C. Then
the solution was removed and the cells were washed once with TBS-D
and once with PBS. The cells were mixed with 5 ml pre-warmed medium
supplemented with 10% serum and 5 .mu.g chloroquine diphosphate and
were incubated 1 hour at 37.degree. C. followed by three times
washes with serum-free medium. Finally, the cells were maintained
in 10 ml culture medium and incubated at 37.degree. C. for 4
days.
[0097] Virus-containing supernatants were collected, filtered
through a 0.45 .mu.g pore-size filter to remove any cells,
aliquoted and kept at -70.degree. C. as virus stock. Titration of
virus was performed by infecting MT-2 cells with serial dilutions
of virus. One hundred microliters from each five-fold serial
dilution of the virus supernatants served as inoculum for 200,000
MT 2 cells. After 16 hours adsorption, the cells were washed five
times and resuspended in 1.5 ml culture medium in 24-well plates
(Costar Corporation). Medium was changed at 4, 7, and 11 days post
infection and p24 production in the supernatants was tested on day
14. The 50% tissue culture infectious dose (TCID.sub.50) end point
was calculated according to the Reed-Muench formula. (Reed and
Muench, Am. J. Hygiene 27: 493-497 (1938)).
[0098] CD4+ T-cell lines MT-2, C91-PL, C8166, CEM, HUT.sub.78, H9,
Jurkat and Molt-3, monocytic cell lines U937, and THP-1, were
propagated and maintained in RPM11640 medium (GIBCO) supplemented
with 10% (v/v) heat-inactivated fetal bovine serum (GIBCO),
penicillin (100 u/ml) and streptomycin (100 u/ml). Hela cells were
grown in medium 199 with Hank's salt supplemented with 2% FBS, 0.8%
dextrose and antibiotics.
[0099] Peripheral blood mononuclear cells (PBMCs) were purified by
Ficoll-Hypaque density gradient centrifugation and stimulated with
phytohemagglutinin (KEBO Lab) for three days in RPMI 1640 medium
supplemented as described above before being infected. Dendritic
cells (DCs) were generated from blood monocytes that were purified
from the mononuclear fraction by adherence to plastic as described
by Rormani et al., J. Exp. Med 180:83-93 (1994). In brief, blood
mononuclear cells were purified, as described above, and adherence
was carried out onto tissue-culture flasks in RPMI medium for two
hours, then the non-adherent cells were washed away with PBS. The
adherent cells were cultured for seven days in medium with GM-CSF
(25 u/ml) and IL-4 (4.5 u/ml) and then were infected with
virus.
[0100] Infections were performed on cell-lines MT-2, C9 1-PL,
C8166, CEM, HUT.sub.78, H9, Jurkat, Molt-3, U937, THP-1, PBMCs and
dendritic cells (DCs). For CD4+ cell-lines, 200,000 cells were
incubated in 37.degree. C. with wild-type or mutant virus at 100
TCID.sub.50 for 16 hours, then washed five times, resuspended in
1.5 ml fresh RPMI medium supplemented with 10% FBS, antibiotics and
Polybrene (Sigma, 2 .mu.g/ml) and incubated in 24-well plate in
37.degree. C. in 5% CO.sub.2 with humidity. For PBMCs, 500,000
cells were used and cultured in RPMI medium supplemented with
proleukin (Eurocetus, 150 u/ml), hydrocortisone (Sigma, 5 .mu.g/ml)
and polybrene (Sigma, 2 .mu.g/ml). For DCs, 800,000 cells were
exposed to virus for 1, 16 and 48 hours, respectively followed by 3
times of wash in PBS and gentle treatment of 0.05% trypsin in
37.degree. C. for 5 minutes to remove any surface-bound virus, as
described by Grannelli-Pipemo et al, J. Exp. Med. 184:2433-2438
(1996). As a control, PBMCs were exposed to mp8 in the same way.
The cells were washed, collected and lysed to isolate DNAs by
phenol/chloroform extraction then a semi-quantitative PCR detecting
LTR sequence was performed. Ten-fold serial dilutions were made on
the DNAs and the LTR PCR was performed in a 40 cycles, using a
three primer "nested" configuration as described before. (Hwang et
al., Science 253: 71-74 (1991)). For DC-PBMC co-culture and DC-MT-2
co-culture experiments, 250,000 DCs were exposed to virus for 16
hours followed by five times of washes until no p24 could be
detected (less than 5 pg/ml). Then the cells were gently treated
with 0.05% trypsin that destroys the HIV-1 binding epitope on CD4
and removes any surface-bound virus. After washing, the cells were
resuspended in RPMI culture medium mixed with 200,000 PBMCs cells
or 100,000 MT-2 cells.
[0101] For infection experiments, culture medium was changed at 4,
7, 11, 14 and 17 days post infection and viral growth was
determined by p24 levels using an HIV-1 p24 ELISA kit (Abbott
Laboratories, North Chicago, USA). The ELISA quantitation of the
p24 assay was used to quantitate the level of p24 in each virus
sample and this assay had a linear dose-response range from 20 pg
to 640 pg of p24 per ml. All virus samples were assayed at multiple
dilutions and p24 amount was determined with the aid of a
regression line. DNAs were isolated from the cells cultured for 17
days post infection and direct sequencing of the V3 region was
performed on these DNAs to verify the mutation.
[0102] Immunocytochemistry was also performed on infected and
uninfected MT-2 cells by an APAAP (Alkaline Phosphatase
Anti-Alkaline Phosphatase immunocomplexes) sandwich technique as
previously described in Kowalski et al., Science 237:1351-1355
(1987). Cells were washed twice in PBS and fixed on slides by
acetone for 15 minutes. Then the cells were incubated in succession
with the primary antibody mouse anti-HIV-1 p24, the secondary
antibody rabbit anti-mouse immunoglobulins, and mouse APAAP
monoclonal antibody (DAKO) for 30 minutes at 37.degree. C.,
respectively, in a humid chamber followed by washing in PBS for 5
minutes. After chromogenic substrate was added and incubated for 20
minutes at room temperature, slides were washed in H.sub.2O,
mounted in glycerol and viewed under microscopy (magnification
.times.100). The monoclonal antibodies (Mabs) mouse anti-HIV-1 p24
(DAKO, diluted 1:20), rabbit anti-mouse immunoglobulins (diluted
1:25) and mouse immunocomplexes of Mab to calf intestinal alkaline
phosphatase and calf intestinal alkaline phosphatase (APAAP,
diluted 1:20) were used for the immunocytochemistry assay.
[0103] Additionally, electron microscopy was performed on infected
cells. Freshly infected cells were fixed on day 7 by 2.5%
glutaraldehyde and postfixed in 1% OSO4. The cells were dehydrated,
embedded with epoxy resins and stained with 1% uranyl acetate. Epon
sections of virus infected cells were made 60-80 nm thin. The
specimens were analyzed in a Zeiss CEM 902 at an accelerating
voltage of 80 kV, which was equipped with a spectrometer to improve
image quality. A liquid nitrogen cooling trap was used to reduce
beam damage.
[0104] In another set of experiments, more evidence that
GPG-NH.sub.2 inhibits HIV-1 infection by a mechanism other than V3
loop inhibition was obtained. Accordingly, the ability of
GPG-NH.sub.2 to inhibit the infectivity of wild-type and V3-loop
deletion mutants (GPG domain) in MT-2 cells was determined. In
these experiments, approximately 200,000 MT-2 cells were infected
with HIV-1.sub.Bru wild-type and GPG-deleted mutants mp8 and mp10
at 25 TCID.sub.50 to test the inhibitory effect of GPG-NH.sub.2 The
MT-2 cells were resuspended in 1 ml of RPMT 1640 medium
supplemented with 10% (v/v) heat-inactivated fetal bovine serum
(FBS, GIBCO), penicillin (100.mu./ml), streptomycin (100.mu./ml)
and Polybrene (Sigma 2 .mu.g/ml) with or without the presence of
GPG-NH.sub.2 at concentration 20 of .mu.M and 100 .mu.M.
Thereafter, viruses were added at 25 TCID.sub.50 in a volume of
20-30 .mu.l. Cells were incubated with virus at 37.degree. C. for
16 hr then loosely pelleted by centrifugation at 170.times.g for 7
minutes. The cells were then washed three times in RPMI medium
without peptides at room temperature by cell sedimentation at
170.times.g for 7 minutes as above. After the final wash, the cells
were resuspended in RPMI culture medium in 24-well plate (Costar
corporation) then kept at 37.degree. C. in 5% CO.sub.2 with
humidity. Culture supernatants were collected when medium was
changed at day 4, 7, 11 and 14 post infection. To monitor the
replication of virus, HIV-1 p24 antigen protein in the supernatants
from day 7 and 14 was assayed by a ELISA kit (Abbott Laboratories)
which has a linear dose-response range from 20 pg to 640 pg of p24
per ml and the p24 amount can be determined with aid of the
regression line.
[0105] The results from the experiments described in this example,
discussed in greater detail below, verify that GPG-NH.sub.2
inhibits HIV-1 infection in a V3 loop independent manner.
[0106] Small peptides inhibit HIV-1 infection in a V3 loop
independent manner
[0107] To determine if the GPG-deletion in the V3 loop affected the
production of virus, the proviral plasmid DNAs (both wild-type and
mutant) were transfected into the CD4 negative cell line Hela, as
well as, the CD4 positive cell lines MT-2 and HUT.sub.78. Culture
supernatants were collected every day and virus production was
monitored by measuring p24 levels. A similar growth pattern was
observed for the wild-type virus (WT) and the mutants from Hela
transfections, within a 6-day time frame. The p24 levels kept
increasing until day 4, then stayed on a plateau. Hence, both the
mutant and the wild-type proviral DNAs were equally well expressed
in these cells. Similar results were obtained from HUT.sub.78,
transfectants and syncytia were observed in these transfected
HUT.sub.78 cells. The pattern of p24 production from MT-2
transfections, however, were notably different from those observed
in Hela and HUT78 cells. The p24 product level kept increasing
beyond day four although the p24 production of cells transfected
with the mutant virus proviral DNAs were lower than those
transfected with the wild-type virus proviral DNA. On day 6 post
transfection, the wild-type produced 1,380 ng/ml of p24 while the
p24 production of mp8 and mp10 were 15.8 ng/ml and 13.7 ng/ml,
respectively. These results demonstrate that GPG-deleted mutant
progeny viruses were produced and could infect non-transfected CD4+
MT-2 cells, albeit apparently not as efficiently as did wild-type
progeny. DNAs from these transfected cells were sequenced and the
GPG deletion was verified for both the mp8 and the mp10
progeny.
[0108] Next, the ability of the mutant molecular clones to generate
virus particles capable of establishing an infection was further
analyzed. Hela cells and MT-2 cells were transfected with the
proviral DNAs and four days post transfection the culture
supernatants were collected, filtered, assayed for p24 levels and
aliquots were frozen at -70.degree. C. as virus stocks. Viral
titration (TCID.sub.50) was performed on MT-2 cells. Supernatants
from MT-2 transfectants, adjusted to contain the same amount of
p24, the wild-type virus yielded 83,300 TCID.sub.50/ml whereas the
mutants, mp8 and mp10 yielded 16,700 and 25,000 TCID.sub.50/ml,
respectively--about five fold less than what was obtained with the
wild-type virus. In concordance with a lower p24 production of the
Hela transfectant supernatants, the titers they yielded were also
much lower, 70 and 10 TCID.sub.50/ml for WT and mutants
respectively. Thus, although the mutant virus was still infectious,
deletion of the GPG motif in V3 may have reduced the viral
virulence in these cells. This was further tested by infection of
MT-2 cells and monitored the production of progeny virus. The virus
stocks from both Hela and MT-2 transfections were then used to
infect MT-2 cells (100 TCID.sub.50 wild type or mutant virus from
MT-2 transfectant supernatants, or five TCID.sub.50 of Virus from
Hela transfectant stocks). The cells were incubated with virus for
16 hours and then washed. Thereafter, the cells were resuspended
and incubated at 37.degree. C. Virus replication was monitored by
measuring p24 levels and cytopathic effects. With virus from MT-2
transfectants, wild-type (WT), as well as, the mutant virus (mp8
and mp10), all showed viral replication by p24 production.
Wild-type reached peak p24 levels of 2,150 ng/ml at day 11 post
infection while the mutant viruses exhibited approximately a 4-day
delay, with peak p24 values of 1,580 ng/ml and 1,760 ng/ml for mp8
and mp10, respectively at day 14 post infection. Infections of MT-2
cells with virus from Hela transfectants (at 5 TCID.sub.50) also
yielded p24 production of both the WT and the mutants with similar
growth kinetics as those obtained with MT-2 cell produced virus.
DNAs were isolated from all infected cells and the mutation was
verified by V3 sequencing, indicating that the growth of the mutant
virus was not due to reversion to or pick up of the wild-type
sequence.
[0109] Syncytium formation was also observed in the MT-2 cells
infected with both the WT and the mutants. Cell cultures were fixed
at day 7 post infection and was used for immunocytochemistry using
the APAAP sandwich technique. The infected cells were immunostained
and gave a red color. Syncytia were observed in both WT and mutant
virus infected MT-2 cells, although WT virus induced syncytia
earlier (4 days post infection) than the mutants (after 6 days).
Electron microscopy (EM) further revealed that the mutant virus
infected MT-2 cells produced HIV-1 particles. HIV-1 particles,
having a characteristic cone-shaped core, were seen. These data
confirmed that the GPG-deletion mutant virus remained infectious in
MT-2 cells.
[0110] Conclusive evidence that GPG-NH2 inhibits viral infection by
a mechanism different than a V3 loop interaction was obtained when
experiments that assessed the ability of GPG-NH.sub.2 to inhibit
the infectivity of wild-type and V3-loop deletion mutants (GPG
domain) in MT-2 cells were performed. At both 7 days and 14 days
after infection, a considerable reduction in wild type and mutant
viral infection was seen. See Table 10. At 20 .mu.M and 100 .mu.M,
GPG-NH.sub.2 effectively reduced reduced wild type infection and
infection mediated by the GPG deletion constructs mp8 and mp10. In
fact, at 7 days post infection and 100 .mu.M GPG-NH2, an equally
complete reduction of viral infectivity was observed for wild-type,
mp8, and mp10. These results established that GPG-NH2 was
inhibiting HIV-1 infection by a mechanism independent from an
interaction with the GPG domain of the V3 loop.
10 TABLE 10 p24 pg/ml GPG control GPG 20 .mu.M reduction % 100
.mu.M reduction % Day 7 WT 33800 23900 29 3390 90 mp8 3170 2420 24
208 93 mp10 3120 1560 50 173 94 Day 14 WT 357000 223000 38 181000
49 mp8 148000 69100 53 7410 95 mp10 470000 51500 89 47700 90
[0111] The data presented herein establish that small peptides
having a modified carboxy terminus inhibit viral infection (e.g.,
HIV-1, HIV-2, and SIV infection), bind to p24, and interrupt proper
capsid assembly. The many assays detailed above can be used to
identify the ability of any small peptide, modified small peptide,
oligopeptide, or peptidomimetic to prevent or inhibit HIV or SIV
infection. Similar techniques can also be used to identify the
ability of any small peptide, modified small peptide, oligopeptide,
or peptidomimetic to prevent or inhibit other viral infections.
[0112] Because the sequence of several viral capsid proteins are
known, the design, manufacture, and identification of small
peptides in amide form that prevent proper assembly of different
viral capsids is straightforward. Several viral capsid proteins,
for instance, contain a 20 amino acid long homology region called
the major homology region (MHR), that exists within the
carboxyl-terminal domain of many onco- and lentiviruses. (See FIG.
5). FIG. 5 shows the carboxyl-terminal domain of HIV-1 (residues
146-231) and compares this sequence to the capsid protein sequences
of other viruses, some of which infect birds, mice, and monkeys.
Notably, considerable homology in the sequences of these viral
capsid proteins is found. Investigators have observed that the
carboxyl-terminal domain is required for capsid dimerization and
viral assembly in HIV-1. (Gamble et al., Science 278: 849 (1997),
herein incorporated by reference in its entirety). While the small
peptides that exhibited antiviral activity in the assays described
in this disclosure fully or partially corresponded to regions of
the carboxyl-terminal domain of HIV-1, HIV-2, or SIV, regions of
the N-terminal domain of viruses are important for capsid assembly
and the design and synthesis of small peptides that either fully or
partially correspond to amino acids of the N-terminal region of
viral capsid proteins are desirable embodiments of the present
invention. The use of small peptides that fully or partially
correspond to amino acids within the MHR region and the
carboxyl-terninal domain of viral capsid proteins, however, are
preferred embodiments of the present invention.
[0113] The inventor has shown several novel small peptides in amide
form that fully or partially correspond to to capsid protein
sequence of three different viruses effectively inhibit and/or
prevent infection by these viruses. The strategies employed herein
can be used to produce additional small peptides in amide form that
fully or partially correspond to the capsid protein sequences of
other viruses. By designing and manufacturing small peptides,
oligopeptides, and/or peptidomimetics that correspond to regions of
the sequences disclosed in FIG. 5, for example, new molecules that
inhibit HIV, SIV, RSV, HTLV-1, MMTV, MPMV, and MMLV infection can
be rapidly identified by using the screening techniques discussed
above or modifications of these assays, as would be apparent to one
of skill in the art. Further, many of the sequences of other viral
capsid proteins are known, such as members of the arenavirus,
rotavirus, orbivirus, retrovirus, papillomavirus, adenovirus,
herpesvirus, paramyxovirus, myxovirus, and hepadnavirus families.
Several small peptides, oligopeptides, and/or peptidomimetics that
fully or partially correspond to these sequences can be selected
and rapidly screened to identify those that effectively inhibit
and/or prevent viral infection by using the viral infectivity
assays and/or electron microscopy techniques described herein, or
modifications of these assays as would be apparent to those of
skill in the art given the present disclosure.
[0114] Desirable embodiments include small peptides (more than one
amino acid and less than or equal to 10 amino acids in length)
having a modified carboxy terminus that are used to interrupt
capsid assembly and inhibit viral infection. Preferably,
dipeptides, tripeptides, and oligopetides and corresponding
peptidomimetics having a sequence that is found in the HIV or SIV
capsid are used. For example, an oligopeptide of the present
invention may have four amino acids, five amino acids, six amino
acids, seven amino acids, eight, or nine or ten amino acids and
peptidomimetics of the present invention may have structures that
resemble four, five, six, seven, eight, nine, or ten amino acids.
These oligopeptides also desirably include the full or partial
sequences found in the tripeptides GPG-NH.sub.2, GKG-NH.sub.2,
CQG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2, ALG-NH.sub.2,
GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2, SLG-NH.sub.2, and
SPT-NH.sub.2. Peptidomimetics that resemble dipeptides, tripeptides
and oligopeptides also, preferably, correspond to a sequence that
is found in GPG-NH.sub.2, GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2,
KQG-NH.sub.2, ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2,
ASG-NH.sub.2, SLG-NH.sub.2, and SPT-NH.sub.2. It is preferred that
the small peptides possess a modulation group (e.g., an amide
group) at their carboxy termini (CO--NH.sub.2) rather than a
carboxyl group (COOH). Small peptides having other modulation
groups at the carboxy terminus, can also be used but desirably, the
attached modulation groups have the same charge and sterically
behave the same as an amide group. (See U.S. Pat. No. 5,627,035 to
Vahlne et al., for an assay to compare peptides having differing
substituents at the carboxyl terminus). In some embodiments, the
addition of an acetyl or methyl group at either end of a small
peptide is desirable so as to improve uptake of the small peptide
or prevent exo-protease digestion or both.
[0115] In the following disclosure, several approaches are provided
to make biotechnological tools and pharmaceutical compositions
comprising dipeptides, tripeptides, oligopeptides of less than or
equal to 10 amino acids, and peptidomimetics that resemble
tripeptides and oligopeptides of less than or equal to 10 amino
acids (collectively referred to as a "peptide agent(s)"). It should
be noted that the term "peptide agents" includes dipeptides,
tripeptides, and oligopeptides of less than equal to 10 amino
acids. "Peptide agents" are, for example, peptides of two, three,
four, five, six, seven, eight, nine, or ten amino acids and
peptidomimetics that resemble peptides of two, three, four, five,
six, seven, eight, nine, or ten amino acids. Further, "peptide
agents" are peptides of two, three, four, five, six, seven, eight,
nine, or ten amino acids or peptidomimetics that resemble two,
three, four, five, six, seven, eight, nine, or ten amino acids that
are provided as multimeric or multimerized agents, as described
below.
[0116] Desirable biotechnological tools or components to
prophylactic or therapeutic agents, provide the peptide agent in
such a form or in such a way that a sufficient affinity or
inhibition of a virus, such as HIV-1, HIV-2, or SIV, is obtained.
While a natural monomeric peptide agent (e.g., appearing as
discrete units of the peptide agent each carrying only one binding
epitope) is sufficient to bind a capsomere protein, such as p24,
and/or interfere with capsid assembly and/or prevent viral
infection, such as HIV-1, HIV-2, or SIV infection, synthetic
ligands or multimeric ligands (e.g., appearing as multiple units of
the peptide agent with several binding epitopes) may have far
greater ability to bind a capsomere protein, such as p24, and/or
interfere with capsid assembly and/or prevent viral infection, such
as HIV-1, HIV-2, or SIV infection. It should be noted that the term
"multimeric" is meant to refer to the presence of more than one
unit of a ligand, for example several individual molecules of a
tripeptide, oligopeptide, or a peptidomimetic, as distinguished
from the term "multimerized" that refers to the presence of more
than one ligand joined as a single discrete unit, for example
several tripeptides, oligopeptides, or peptidomimetic molecules
joined in tandem.
[0117] Preparation of multimeric supports and multimerized
ligands
[0118] A multimeric agent (synthetic or natural) that binds a
capsomere protein, such as p24, and/or interferes with capsid
assembly and/or inhibits viral infection, such as HIV-1, HIV-2, or
SIV infection, may be obtained by coupling a tripeptide,
oligopeptide or a peptidomimetic to a macromolecular support. The
term "support" as used herein includes a carrier, a resin or any
macromolecular structure used to attach, immobilize, or stabilize a
peptide agent. Solid supports include, but are not limited to, the
walls of wells of a reaction tray, test tubes, polystyrene beads,
magnetic beads, nitrocellulose strips, membranes, microparticles
such as latex particles, sheep (or other animal) red blood cells,
artificial cells and others. The term Support also includes
carriers as that term is understood for the preparation of
pharmaceuticals.
[0119] The macromolecular support may have a hydrophobic surface
that interacts with a portion of the peptide agent by hydrophobic
non-covalent interaction. The hydrophobic surface of the support
may also be a polymer such as plastic or any other polymer in which
hydrophobic groups have been linked such as polystyrene,
polyethylene or polyvinyl. Alternatively, the peptide agent can be
covalently bound to carriers including proteins and
oligo/polysaccarides (e.g. cellulose, starch, glycogen, chitosane
or aminated sepharose). In these later embodiments, a reactive
group on the peptide agent, such as a hydroxy or an amino group,
may be used to join to a reactive group on the carrier so as to
create the covalent bond. The support may also have a charged
surface that interacts with the peptide agent. Additionally, the
support may have other reactive groups that can be chemically
activated so as to attach a peptide agent. For example, cyanogen
bromide activated matrices, epoxy activated matrices, thio and
thiopropyl gels, nitrophenyl chloroformate and N-hydroxy
succinimide chlorformate linkages, and oxirane acrylic supports are
common in the art.
[0120] The support may also comprise an inorganic carrier such as
silicon oxide material (e.g. silica gel, zeolite, diatomaceous
earth or aminated glass) to which the peptide agent is covalently
linked through a hydroxy, carboxy or amino group and a reactive
group on the carrier. Furthermore, in some embodiments, a liposome
or lipid bilayer (natural or synthetic) is contemplated as a
support and peptide agents are attached to the membrane surface or
are incorporated into the membrane by techniques in liposome
engineering. By one approach, liposome multimeric supports comprise
a peptide agent that is exposed on the surface of the bilayer and a
second domain that anchors the peptide agent to the lipid bilayer.
The anchor may be constructed of hydrophobic amino acid residues,
resembling known transmembrane domains, or may comprise ceramides
that are attached to the first domain by conventional
techniques.
[0121] Supports or carriers for use in the body, (i.e. for
prophylactic or therapeutic applications) are desirably
physiological, non-toxic and preferably, non-immunoresponsive.
Contemplated carriers for use in the body include poly-L-lysine,
poly-D, L-alanine, liposomes, and Chromosorb.RTM. (Johns-Manville
Products, Denver Colo.). Ligand conjugated Chromosorb.RTM.
(Synsorb-Pk) has been tested in humans for the prevention of
hemolytic-uremic syndrome and was reported as not presenting
adverse reactions. (Armstrong et al. J. Infectious Diseases,
171:1042-1045 (1995)). For some embodiments, the present inventor
contemplates the administration of a "naked" carrier (i.e., lacking
an attached peptide agent) that has the capacity to attach a
peptide agent in the body of a subject. By this approach, a
"prodrug-type" therapy is envisioned in which the naked carrier is
administered separately from the peptide agent and, once both are
in the body of the subject, the carrier and the peptide agent are
assembled into a multimeric complex.
[0122] The insertion of linkers, such as .lambda. linkers, of an
appropriate length between the peptide agent and the support are
also contemplated so as to encourage greater flexibility of the
peptide agent and thereby overcome any steric hindrance that may be
presented by the support. The determination of an appropriate
length of linker that allows for optimal binding to a capsomere
protein, such as p24, and/or interference with capsid assembly
and/or inhibition of viral infection, such as HIV or SIV infection,
can be determined by screening the peptide agents with varying
linkers in the assays detailed in the present disclosure.
[0123] A composite support comprising more than one type of peptide
agent is also an embodiment. A "composite support" may be a
carrier, a resin, or any macromolecular structure used to attach or
immobilize two or more different peptide agents that bind to a
capsomere protein, such as p24, and/or interfere with capsid
assembly and/or inhibit viral infection, such as HIV or SIV
infection. In some embodiments, a liposome or lipid bilayer
(natural or synthetic) is contemplated for use in constructing a
composite support and peptide agents are attached to the membrane
surface or are incorporated into the membrane using techniques in
liposome engineering.
[0124] As above, the insertion of linkers, such as .lambda.
linkers, of an appropriate length between the peptide agent and the
support is also contemplated so as to encourage greater flexibility
in the molecule and thereby overcome any steric hindrance that may
occur. The determination of an appropriate length of linker that
allows for optimal binding to a capsomere protein, such as p24,
and/or interference with capsid assembly and/or inhibition of viral
infection, such as HIV or SIV infection, can be determined by
screening the ligands with varying linkers in the assays detailed
in the present disclosure.
[0125] In other embodiments of the present invention, the
multimeric and composite supports discussed above may have attached
multimerized ligands so as to create a "multimerized-multimeric
support" and a "multimerized-composite support", respectively. A
multimerized ligand may, for example, be obtained by coupling two
or more peptide agents in tandem using conventional techniques in
molecular biology. The multimerized form of the ligand may be
advantageous for many applications because of the ability to obtain
an agent with a better ability to bind to a capsomere protein, such
as p24, and/or interfere with capsid assembly and/or inhibit viral
infection, such as HIV or SIV infection. Further, the incorporation
of linkers or spacers, such as flexible .lambda. linkers, between
the individual domains that make-up the multimerized agent is an
advantageous embodiment. The insertion of .lambda. linkers of an
appropriate length between protein binding domains, for example,
may encourage greater flexibility in the molecule and can overcome
steric hindrance. Similarly, the insertion of linkers between the
multimerized ligand and the support may encourage greater
flexibility and limit steric hindrance presented by the support.
The determination of an appropriate length of linker that allows
for optimal binding to p24 and/or interference with capsid assembly
and/or inhibition of HIV or SIV infection, can be determined by
screening the ligands with varying linkers in the assays detailed
in this disclosure.
[0126] In preferable embodiments, the various types of supports
discussed above are created using the tripeptides GPG-NH.sub.2,
GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2, KQG-NH.sub.2,
ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2, ASG-NH.sub.2,
SLG-NH.sub.2, and SPT-NH.sub.2. The multimeric supports, composite
supports, multimerized-multimeric supports, or
multimerized-composite supports, collectively referred to as
"support-bound agents", are also preferably constructed using the
tripeptides GPG-NH.sub.2, GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2,
KQG-NH.sub.2, ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2,
ASG-NH.sub.2, SLG-NH.sub.2, and SPT-NH.sub.2.
[0127] In the discussion below, several embodiments of the
invention that have therapeutic and/ or prophylactic application
are described.
[0128] Therapeutic and prophylactic applications
[0129] The monomeric and multimeric peptide agents described herein
are suitable for treatment of subjects either as a preventive
measure to avoid viral infections, such as HIV or SIV infection, or
as a therapeutic to treat subjects already infected with a virus,
such as HIV or SIV. Although anyone could be treated with the
peptides as a prophylactic, the most suitable subjects are people
at risk for viral infection. Such subjects include, but are not
limited to, homosexuals, prostitutes, intravenous drug users,
hemophiliacs, children born to virus-infected mothers, and those in
the medical profession who have contact with patients or biological
samples.
[0130] The pharmacologically active compounds of this invention can
be processed in accordance with conventional methods of galenic
pharmacy to produce medicinal agents for administration to
patients, e.g., mammals including humans. The peptide agents can be
incorporated into a pharmaceutical product with and without
modification. Further, the manufacture of pharmaceuticals or
therapeutic agents that deliver the peptide agent or a nucleic acid
sequence encoding a small peptide by several routes is an
embodiment. For example, and not by way of limitation, DNA, RNA,
and viral vectors having sequence encoding a small peptide that
inhibits viral replication by interupting capsid assembly are
contemplated. Nucleic acids encoding a desired peptide agent can be
administered alone or in combination with peptide agents.
[0131] The compounds described herein can be employed in admixture
with conventional excipients, i.e., pharmaceutically acceptable
organic or inorganic carrier substances suitable for parenteral,
enteral (e.g., oral) or topical application that do not
deleteriously react with the peptide agents. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, gum arabic, vegetable oils,
benzyl alcohols, polyetylene glycols, gelatine, carbohydrates such
as lactose, amylose or starch, magnesium stearate, talc, silicic
acid, viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, pentaerythritol fatty acid esters, hydroxy
methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical
preparations can be sterilized and if desired mixed with auxiliary
agents, e.g., lubricants, preservatives, stabilizers, wetting
agents, emulsifiers, salts for influencing osmotic pressure,
buffers, coloring, flavoring and/or aromatic substances and the
like that do not deleteriously react with the active compounds.
They can also be combined where desired with other active agents,
e.g., vitamins.
[0132] In some embodiments, therapeutic agents comprising peptide
agents are administered in conjunction with other therapeutic
agents that treat viral infections, such as HIV infection, so as to
achieve a better viral response. At present four different classes
of drugs are in clinical use in the antiviral treatment of HIV-1
infection in humans. These are (i) nucleoside analogue reverse
transcriptase inhibitors (NRTIs), such as zidovidine, lamivudine,
stavudine, didanosine, abacavir, and zalcitabine; (ii) nucleotide
analogue reverse transcriptase inhibitors, such as adetovir and
pivaxir; (iii) non-nucleoside reverse transcriptase inhibitors
(NNRTIs), such as efavirenz, nevirapine, and delavirdine; and (iv)
protease inhibitors, such as indinavir, nelfinavir, ritonavir,
saquinavir and amprenavir. By simultaneously using two, three, or
four different classes of drugs in conjunction with administration
of the peptide agents, HIV is less likely to develop resistance,
since it is less probable that multiple mutations that overcome the
different classes of drugs and the peptide agents will appear in
the same virus particle.
[0133] It is thus a preferred embodiment of the present invention
that peptide agents be given in combination with nucleoside
analogue reverse transcriptase inhibitors, nucleotide analogue
reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and protease inhibitors at doses and by
methods known to those of skill in the art. Medicaments comprising
the peptide agents and nucleoside analogue reverse transcriptase
inhibitors, nucleotide analogue reverse transcriptase inhibitors,
non-nucleoside reverse transcriptase inhibitors, and protease
inhibitors are also embodiments of the present invention.
[0134] Studies on the efficacy of treatment of HIV infection with
combinations of GPG-NH.sub.2 and conventional antiviral agents can
be found in the example provided below.
EXAMPLE 4
[0135] In this example, experiments are presented in which
different combinations of a small peptide in amide form and AZT
were tested to determine whether the two compounds could complement
one another to inhibit HIV-1 replication. (See FIG. 6).
Accordingly, 200,000 HUT.sub.78 cells were infected with HIV-1 SF-2
virus at 25 TCID.sub.50, with or without the presence of different
concentrations of GPG-NH.sub.2, AZT or Ritonavir ("Rito") and
combinations of these compounds. The numbers shown in FIG. 6
represent micromolar concentrations of the inhibiting compounds.
Cells were incubated with virus at 37.degree. C. for 1 hr with the
various inhibitors and were subsequently washed three times. Next,
the cells were resuspended in RPMI 1640 medium containing the
antiviral agent and/or the peptides supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (FBS, GIBCO), penicillin (100
u/ml), streptomycin (100 u/ml) and Polybrene (Sigma, 2 .mu.g/ml)
and cultured in 24-well plate (Costar corporation) at 37.degree. C.
in 5% CO.sub.2 with humidity. Culture supernatants were collected
every four days and medium was changed until day 14 post infection.
To monitor the replication of virus, HIV-1 p24 antigen protein in
the supernatants was assayed using a commercially available kit
(Abbott).
[0136] It was observed that GPG-NH.sub.2 enhanced the inhibition of
the replication of HIV-1 in the presence of AZT synergistically,
whereas, the small peptide only exhibited an additive antiviral
effect to that of the protease inhibitor Ritonavir. Nevertheless,
these experiments validate data presented above that small peptides
inhibit HIV-1 by a mechanism apart from the manner in which
nucleoside analogs and protease inhibitors interfere with viral
replication. Further, these experiments demonstrate that a novel
treatment protocol for HIV-1 infection comprising small peptides
and AZT and/or Ritonavir is efficacious.
[0137] In the following disclosure, doses and methods of
administration are provided.
[0138] Dosage and methods of administration
[0139] The effective dose and method of administration of a
particular peptide agent formulation may vary based on the
individual patient and the stage of the disease, as well as other
factors known to those of skill in the art. Therapeutic efficacy
and toxicity of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., ED50 (the dose therapeutically effective in 50% of the
population) and LD50 (the dose lethal to 50% of the population).
The dose ratio of toxic to therapeutic effects is the therapeutic
index, and it can be expressed as the ratio, LD50/ED50.
Pharmaceutical compositions that exhibit large therapeutic indices
are preferred. The data obtained from cell culture assays and
animal studies is used in formulating a range of dosage for human
use. The dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.
[0140] The exact dosage is chosen by the individual physician in
view of the patient to be treated. Dosage and administration are
adjusted to provide sufficient levels of the active moiety or to
maintain the desired effect. Additional factors that may be taken
into account include the severity of the disease state, age, weight
and gender of the patient; diet, time and frequency of
administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Short acting pharmaceutical
compositions are administered daily whereas long acting
pharmaceutical compositions are administered every 2, 3 to 4 days,
every week, or once every two weeks. Depending on half-life and
clearance rate of the particular formulation, the pharmaceutical
compositions of the invention are administered once, twice, three,
four, five, six, seven, eight, nine, ten or more times per day.
[0141] Normal dosage amounts may vary from approximately 1 to
100,000 micrograms, up to a total dose of about 10 grams, depending
upon the route of administration. Desirable dosages include 250
.mu.g, 500 .mu.g, 1 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300
mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg,
750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g,
1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 3 g, 4 g, 5, 6 g, 7 g, 8 g,
9 g, and 10 g. Additionally, the concentrations of the peptide
agents can be quite high in embodiments that administer the agents
in a topical form. Molar concentrations of peptide agents can be
used with some embodiments. Desirable concentrations for topical
administration and/or for coating medical equipment range from 100
.mu.M to 800 mM. Preferable concentrations for these embodiments
range from 500 .mu.M to 500 mM. For example, preferred
concentrations for use in topical applications and/or for coating
medical equipment include 500 .mu.M, 550 .mu.M, 600 .mu.M, 650
.mu.M, 700 .mu.M, 750 .mu.M, 800 .mu.M, 850 .mu.M, 900 .mu.M, 1 mM,
5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50
mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150
mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 300 mM, 325 mM, 350 mM,
375 mM, 400 mM, 425 mM, 450 mM, 475 mM, and 500 mM. Guidance as to
particular dosages and methods of delivery is provided in the
literature, (see e.g., U.S. Pat. Nos. 4,657,760; 5,206,344; or
5,225,212) and below.
[0142] More specifically, the dosage of the peptide agents
described herein is one that provides sufficient peptide agent to
attain a desirable effect including binding of a capsomere protein,
such as p24, and/or interference with capsid assembly and/or
inhibition of viral infection, such as HIV and SIV infection.
Accordingly, the dose of peptide agent preferably produces a tissue
or blood concentration or both from approximately 0.1 .mu.M to 500
mM. Desirable doses produce a tissue or blood concentration or both
of about 1 to 800 .mu.M. Preferable doses produce a tissue or blood
concentration of greater than about 10 .mu.M to about 500 .mu.M.
Preferable doses are, for example, the amount of small peptide
required to achieve a tissue or blood concentration or both of 10
.mu.M, 15 .mu.M, 20 .mu.M, 25 .mu.M, 30 .mu.M, 35 .mu.M, 40 .mu.M,
45 .mu.M, 50 .mu.M, 55 .mu.M, 60 .mu.M, 65 .mu.M, 70 .mu.M, 75
.mu.M, 80 .mu.M, 85 .mu.M, 90 .mu.M, 95 .mu.M, 100 .mu.M, 110
.mu.M, 120 .mu.M, 130 .mu.M, 140 .mu.M, 145 .mu.M, 150 .mu.M, 160
.mu.M, 170 .mu.M, 180 .mu.M, 190 .mu.M, 200 .mu.M, 220 .mu.M, 240
.mu.M, 250 .mu.M, 260 .mu.M, 280 .mu.M, 300 .mu.M, 320 .mu.M, 340
.mu.M, 360 .mu.M, 380 .mu.M, 400 .mu.M, 420 .mu.M, 440 .mu.M, 460
.mu.M, 480 .mu.M, and 500 .mu.M. Although doses that produce a
tissue concentration of greater than 800 .mu.M are not preferred,
they can be used with some embodiments of the present invention. A
constant infusion of the peptide can also be provided so as to
maintain a stable concentration in the tissues as measured by blood
levels.
[0143] Higher tissue concentrations can be maintained without harm
due to the low toxicity of the peptides. Attempts to select small
peptide resistant strains of HIV-1 (e.g., GPG-NH.sub.2 resistant
strains) have so far been unsuccessful. The HIV-1 strain HTLV-IIIB
was passaged in the presence of serial dilutions of GPG-NH.sub.2
(limiting dilutions) for more than six months without overt signs
of development of resistance in vitro.
[0144] Routes of administration of the peptide agents include, but
are not limited to, topical, transdermal, parenteral,
gastrointestinal, transbronchial, and transalveolar. Topical
administration is accomplished via a topically applied cream, gel,
rinse, etc. containing a peptide. Transdermal administration is
accomplished by application of a cream, rinse, gel, etc. capable of
allowing the peptide agent to penetrate the skin and enter the
blood stream. Parenteral routes of administration include, but are
not limited to, electrical or direct injection such as direct
injection into a central venous line, intravenous, intramuscular,
intraperitoneal or subcutaneous injection. Gastrointestinal routes
of administration include, but are not limited to, ingestion and
rectal. Transbronchial and transalveolar routes of administration
include, but are not limited to, inhalation, either via the mouth
or intranasally.
[0145] Compositions of peptide agent-containing compounds suitable
for topical application include, but not limited to,
physiologically acceptable implants, ointments, creams, rinses, and
gels. Any liquid, gel, or solid, pharmaceutically acceptable base
in which the peptides are at least minimally soluble is suitable
for topical use in the present invention. Compositions for topical
application are particularly useful during sexual intercourse to
prevent transmission of HIV. Suitable compositions for such use
include, but are not limited to, vaginal or anal suppositories,
creams, and douches.
[0146] Compositions of the peptide agents suitable for transdermal
administration include, but are not limited to, pharmaceutically
acceptable suspensions, oils, creams, and ointments applied
directly to the skin or incorporated into a protective carrier such
as a transdermal device ("transdermal patch"). Examples of suitable
creams, ointments, etc. can be found, for instance, in the
Physician's Desk Reference and are well known in the art. Examples
of suitable transdermal devices are described, for instance, in
U.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen, et al.,
herein incorporated by reference.
[0147] Compositions of the peptide agents suitable for parenteral
administration include, but are not limited to, phannaceutically
acceptable sterile isotonic solutions. Such solutions include, but
are not limited to, saline and phosphate buffered saline for
injection into a central venous line, intravenous, intramuscular,
intraperitoneal, or subcutaneous injection of the peptide
agents.
[0148] Compositions of the peptide agents suitable for
transbronchial and transalveolar administration include, but not
limited to, various types of aerosols for inhalation. For instance,
pentamidine is administered intranasally via aerosol to AIDS
patients to prevent pneumonia caused by pneumocystis carinii.
Devices suitable for transbronchial and transalveolar
administration of the peptides are also embodiments. Such devices
include, but are not limited to, atomizers and vaporizers. Many
forms of currently available atomizers and vaporizers can be
readily adapted to deliver peptide agents.
[0149] Compositions of the peptide agents suitable for
gastrointestinal administration include, but not limited to,
pharmaceutically acceptable powders, pills or liquids for ingestion
and suppositories for rectal administration. Due to the most common
routes of HIV infection and the ease of use, gastrointestinal
administration, particularly oral, is the preferred embodiment of
the present invention. Five-hundred milligram capsules having a
tripeptide (GPG-NH.sub.2) have been prepared and were found to be
stable for a minimum of 12 months when stored at 4.degree. C. As
previously shown in other virus-host systems, specific antiviral
activity of small peptides can be detected in serum after oral
administration. (Miller et al., Appl. Microbiol., 16:1489 (1968)).
Since small peptides apparently evade degradation by the patient's
digestive system, they are ideal for oral administration.
[0150] The peptide agents are also suitable for use in situations
where prevention of HIV infection is important. For instances,
medical personnel are constantly exposed to patients who may be HIV
positive and whose secretions and body fluids contain the HIV
virus. Further, the peptide agents can be formulated into antiviral
compositions for use during sexual intercourse so as to prevent
transmission of HIV. Such compositions are known in the art and
also described in international application published under the PCT
publication number WO90/04390 on May 3, 1990 to Modak et al., which
is incorporated herein by referencein its entirety.
[0151] Aspects of the invention also include a coating for medical
equipment such as gloves, sheets, and work surfaces that protects
against HIV transmission. Alternatively, the peptide agents can be
impregnated into a polymeric medical device. Particularly preferred
are coatings for medical gloves and condoms. Coatings suitable for
use in medical devices can be provided by a powder containing the
peptides or by polymeric coating into which the peptide agents are
suspended. Suitable polymeric materials for coatings or devices are
those that are physiologically acceptable and through which a
therapeutically effective amount of the peptide agent can diffuse.
Suitable polymers include, but are not limited to, polyurethane,
polymethacrylate, polyamide, polyester, polyethylene,
polypropylene, polystyrene, polytetrafluoroethylene,
polyvinyl-chloride, cellulose acetate, silicone elastomers,
collagen, silk, etc. Such coatings are described, for instance, in
U.S. Pat. No. 4,612,337, issued Sep. 16, 1986 to Fox et al. that is
incorporated herein by reference in its entirety.
[0152] In the disclosure below, several toxicological studies on
small peptides are provided and approaches to test the toxicity of
other peptide agents are disclosed.
[0153] Small Peptide Toxicology Studies
[0154] Several toxicology studies on GPG-NH.sub.2 have been
performed and these assays can be reproduced using many more
peptides (e.g., GKG-NH.sub.2, CQG-NH.sub.2, RQG-NH.sub.2,
KQG-NH.sub.2, ALG-NH.sub.2, GVG-NH.sub.2, VGG-NH.sub.2,
ASG-NH.sub.2, SLG-NH.sub.2, and SPT-NH.sub.2). (See e.g., U.S. Pat.
No. 5,627,035 to Vahlne et al.). The effects of GPG-NH.sub.2 on
cultured lymphocytes is provided in the following example.
EXAMPLE 5
[0155] In this example, several toxicology studies that addressed
the effects of small peptides on cultured lymphocytes are
presented. Generally, none of the peptides used in the virus
inhibition assays for HIV-1 tested at concentrations up to 2 mM
exhibited any toxic effect on the cells used, as determined by
their morphological appearance, trypan blue staining and cell
growth. The toxicity of GPG-NH.sub.2 was further tested in (i) a
plaque reduction assay and (ii) a yield inhibition assay of HSV-1.
GPG-NH.sub.2 failed to reduce HSV-1 replication at concentrations
up to 1 mM that not only confirmed its lack of toxicity but also
demonstrated that the small peptide selectively inhibits
HIV-related viruses. This view was reinforced by the observation
that GPG-NH.sub.2 also failed inhibit either influenza or polio
viruses.
[0156] Next, the toxicity to normal human lymphomonocytic cells
(PBMC) was examined. At does as high as 2 mM and exposure as long
as 4 days, GPG-NH.sub.2 did not appreciably affect the viability of
cultured monocytes and lymphocytes. The effect of GPG-NH.sub.2 on
in vitro proliferative response of human mononuclear cells was also
tested. GPG-NH.sub.2 exerted marginal if any direct
anti-proliferative properties on T cells and did not affect the
function of accessory cells (e.g. monocytes and dendritic cells) at
a dose of 20 .mu.M.
[0157] The in vitro 50% cytotoxic concentrations (CC.sub.50) of
various cell lines was also assessed. Accordingly, different
concentrations of GPG-NH.sub.2, up to 40 mM, was supplemented into
the medium culture of T-cell lines HUT.sub.78, H9, CEM-SS, MT-2 and
(non) induced ACH-2, as well as, the macrophage derived cell lines
Jurkat-tat III, THP-1 and (non) induced U-1 cells. After 3 days of
incubation, the number of cells were counted. Trypan-blue dye
exclusion and cell counting of HUT78, MT-2 and Jurkat-tat III
showed about a 40% inhibition of cell growth for 3 days of culture
in the presence of 40 mM GPG-NH2. H9, CEM-SS, and THP-1 showed a
less than 20% decrease in cell number at this GPG-NH2
concentration. Therefore, the ratio of CC.sub.50/IC.sub.50 in vitro
is >10.sup.4. The toxicology experiments presented above prove
that small peptides are of low toxicity to lymphocytes even at high
concentrations.
[0158] The effects of large doses of small peptide on rodents was
also analyzed and these experiments are presented in the next
example.
EXAMPLE 6
[0159] In this example, the results from several in vivo toxicology
studies on small peptides administered in large doses to rodents
are presented. In a first experiment, a large single dose of small
peptide was delivered to mice and the toxic effects were analyzed.
An intravenous injection of GPG-NH.sub.2 in the tail vein of adult
mice at concentrations of up to 1 g per kilo bodyweight gave no
apparent toxic effects to the animals. In a second experiment,
several doses of varying amounts of a small peptide were delivered
to mice for almost three weeks. Groups of mice (five mice in each
group) were given intraperitoneal injections of GPG-NH.sub.2 (0.01,
0.1 and 1 g per kilo bodyweight and day, respectively) starting at
an age of 6 days. Daily injections were continued for 18 days.
Compared to controls, there was no significant influence of
GPG-NH.sub.2 on the mice. In a similar experiment, a four week
toxicology study of GPG-NH.sub.2 was performed by Scantox A/S in
Denmark. GPG-NH.sub.2 was given orally to rats at doses up to 1 g
per kilo bodyweight per day for twenty eight days. No signs of
toxicity to the animals was observed. These in vivo toxicology
experiments prove that the small peptides described herein are of
low toxicity to mammals and can be safely provided at large
doses.
[0160] In further studies, the stability of small peptides in human
blood and plasma was analyzed. The example below discloses these
experiments.
EXAMPLE 7
[0161] This example describes several studies that were performed
to access the stability of small peptides in human blood and
plasma. Accordingly, human blood was taken freshly and treated with
EDTA. Plasma was separated by centrifuging at 2,500 rpm for 20
minutes. GPG-NH.sub.2 was added into blood or plasma at
concentrations of 10 mM or 50 mM followed by incubation in
37.degree. C. for 1, 2 and 4 hours, respectively. As a control,
GPG-NH.sub.2 was added into RPMI 1640 medium supplemented with 10%
(v/v) heat-inactivated fetal bovine serum (FBS, GIBCO), penicillin
(100 u/ml), streptomycin (100 u/ml) and Polybrene (Sigma, 2 g/ml)
and incubated in the same way. After incubation in 37.degree. C.,
the GPG-NH.sub.2 containing blood was centrifuged at 2,500 rpm for
20 minutes to isolate plasma. Some of the plasma samples were
treated with CaCl.sub.2 at concentration of 5 mM in 37.degree. C.
for 10 minutes followed by centrifuging at 13,000 rpm for 30
minutes and the supernatant are referred to as CaCl.sub.2 treated
plasma. Then all the GPG-NH.sub.2 containing plasma samples were
diluted in RPMI medium to give the final concentrations 20 .mu.M or
100 .mu.M of GPG-NH.sub.2 (500 fold dilutions) and were then used
in HIV-1 replication assays.
[0162] The replication assays were performed on HUT.sub.78 cells
and the HIV-1 SF-2 virus strain was used. Briefly, approximately
200,000 cells were resuspended in the diluted GPG-amide containing
medium, plasma, or plasma from blood. The GPG-NH.sub.2 containing
medium, plasma, or plasma from blood was incubated with the cells
for either 1 hr, 2 hr, or 4 hr at 37.degree. C. Subsequently, SF-2
virus was added at 25 TCID.sub.50. After adsorption of 1 hour,
cells were washed three times in RPMI medium then resuspended in
the proper GPG-amide containing plasma and incubated at 37.degree.
C. in 5% CO.sub.2 with humidity. Culture supernatants were
collected every four days and medium was changed until day 14 post
infection. To monitor the replication of virus, HIV-1 p24 antigen
protein in the supernatants was assayed using a commercially
available kit (Abbott). The HIV-1 p24 assay was performed on the
supernatants from day 7 and day 14 post infection. No significant
difference in the ability to inhibit HIV-1 with GPG-NH.sub.2
supplemented medium, plasma, or plasma from blood was observed.
[0163] The stability of GPG-NH.sub.2 in human plasma was also
assessed chemically by thin layer chromatography (TLC). (See FIG.
7). In this experiment, .sup.14C GPG-NH.sub.2 was incubated with
human plasma at 37.degree. C. for 30 minutes, two hours, or eight
hours and then the proteins were separated by TLC and were
visualized by exposure of the chromatograph to autoradiography
film. As shown in FIG. 7 (lanes Hp0, Hp0.5, Hp2, and Hp8), a slight
shift in mobility of the small peptide was observed. Although the
mobility of the small peptide increased somewhat after 30 minutes
of incubation in the human plasma at 37.degree. C., no further
change in mobility was observed at up to 8 hours. Mass-spectrometry
analysis (electrospray analysis) of the TLC spots verified that all
spots, including the spot with increased mobility, were
GPG-NH.sub.2. The experiments above prove that the small peptides
described herein are stable in human blood and plasma; they retain
their antiviral properties, and are not degraded by plasma
proteinases.
[0164] In the disclosure below, several studies on the adsorption,
distribution, and metabolism of the small peptides are
provided.
[0165] Adsorption, distribution, and metabolism of small
peptides
[0166] Because an oral administration of a pharmaceutical
comprising a small peptide is desired, the acid stability of small
peptides was assessed by incubating .sup.14C-labelled GPG-NH.sub.2
in a solution of 50 mM KCl and 0.1M HCl for various time periods.
After acid hydrolysis, the radiolabeled tripeptide was analyzed by
thin layer chromatography (TLC) and the HPTLC plate was developed
with 25% methanol: 25%isopropanol: 15% butanol: 35% (0.1N HOAc and
0.1N NaOAc). As can be seen in FIG. 8, incubation of the tripeptide
for up to 24 hours did not affect the mobility, and hence not the
molecular structure, of the GPG-NH.sub.2. This result established
that small peptides survive acidic conditions similar to that found
in the stomach.
[0167] Additionally, the in vitro uptake of small peptides from the
culture medium into the cells was studied in a series of
experiments. Accordingly, HUT.sub.78, Jurkat-tat III, and MT-2
cells were incubated with .sup.14C-labelled GPG-NH.sub.2 165 nCi
(equivalent to 0.7 .mu.M of GPG-NH.sub.2). An uptake of the small
peptide was observed and between 8% (Jurkat-tat III cells) and 20%
(HUT.sub.78 cells) of the GPG-NH.sub.2 was incorporated in the
cells. This result proved that the incorporation of small peptides
into several cell types was effective.
[0168] Further, the in vivo uptake of small peptides was analyzed
in rats. Rats were fed .sup.14C GPG-NH.sub.2 and, after various
time points, blood, urine, and tissue samples were collected from
the animals. Samples of rat urine taken eight hours after feeding
and rat plasma taken 30 minutes, two hours, and eight hours after
feeding were separated on a TLC plate, as shown in FIG. 7
(designated urine8, rp0.5, rp2, and rp.sup.8, accordingly). All the
tissues were kept in -20.degree. C. after necropsy before any assay
was performed. Ten to thirty micrograms of the tissue samples were
collected from three different locations of each organ analyzed and
the organ tissue was subsequently dissolved in 250 .mu.l of tissue
solublizer (OptiSolv, LKB-Wallac) at 45.degree. C. for four to six
hours. The homogenized tissue solutions were decolorized by the
addition of 50 .mu.l of 30% H.sub.2O.sub.2 and 100 .mu.l of
isopropanol before 3 ml of 0.05M HCl acidified scintillation
cocktail (Luma Gel, Lumac/3M) was added. The radioactivity was
determined with a beta scintillation counter (LKB-Wallac 1218 Rack
Beta). The free/unbound GPG-NH.sub.2 in the plasma was collected by
precipitation with one part of plasma and two parts of ethanol
followed by incubation at -70.degree. C. for one hour and
centrifugation at 20,000.times.g for 15 min at 4.degree. C. Blood
cell samples were diluted with PBS (in the ratio of 2 parts of
blood cells to 1 part of PBS) before 10 .mu.l of the mixture was
sampled and analyzed as the tissue samples. Five to 20 .mu.l of
plasma and urine samples were directly mixed with 3 ml of Ready
Safe (Beckman) fluid before quantification. The results giving the
distribution and the basis for calculation of maximum uptake are
shown in Tables 10 and 11. The values are expressed in nCi.
11TABLE 11 Animal number* Ci/ml or g tissue 1 2 3 4 5 avg Brain 48
34 31 30. 33 35 Kidneys 450 491 467 446 477 466 Liver 599 413 454
507 503 495 Spleen 383 428 414 195 413 366 Thymus 366 288 290 338
372 331 Blood cells 90 81 99 95 101 93 Plasma 140 126 121 142 124
130 Urine 5,846 5,068 6,841 4,557 5,986 5,660 Total urine 7,016
7,096 3,284 283 2,395 4,015 *Animals were sacrificed after 4
hours.
[0169]
12TABLE 12 Animal number* 6 7 8 9 10 avg Brain 43 27 28 25 29 30
Kidneys 255 488 461 401 468 415 Liver 537 419 478 458 578 494
Spleen 307 281 257 336 285 293 Thymus 340 350 323 349 311 334 Blood
51 72 87 71 108 78 cells Plasma 138 123 135 129 148 135 Urine 2,992
5,040 3,121 4,297 2,175 3,525 Total 6,463 6,653 11,173 10,227 7,613
8,426 urine *Animals sacrificed after 8 hours.
[0170] The calculation of the maximum uptake was determined as
follows. The total feeding was 800 .mu.Ci/kg rat body weight (160
.mu.Ci in total per animal). On the assumption that the
GPG-NH.sub.2 and its metabolities were evenly distributed in the
body, the GPG-NH.sub.2 present in tissues would be the average
counts/g from different tissues from the total number of animals
studied divided by the total number of animals and multiplied by
the body weight and the factor 0.9. This factor is derived to omit
the blood volume since an estimate of blood volume is roughly 10%
of the average body weight. For example (from the data of animals
1-5), if the total body weight for five rats was 207 g and the
radioactivity detected from various tissues was
(35+466+495+366+331) or 1,693 nCi and the radioactivity detected
from blood was (93+130) or 223 nCi and the radioactivity detected
in urine was 4, 015 nCi, the maximal intake of the small peptide
can be calculated as:
13 tissue: (35 + 466 + 495 + 366 + 331)/5 * 207 * 0.9 = 63,240 nCi
fluids: blood (93 + 130) * 207 * 0.1 = 4,642 nCi urine: 4,015 nCi
Sum: (63.24 + 4.642 + 4.015)/800 * 0.207 = 0.4342, or 43% maximal
uptake
[0171] The relative distribution of retained/immobilized
GPG-NH.sub.2 and its metabolites in sampled tissues was observed to
be highest in the liver followed by the kidney, followed by the
spleen, followed by the thymus, followed by the brain. The
radioactivity in the urine was observed to double between hours 4
and 8. Mass spectrometric data (electrospray mass spectrometry) of
the urine radioactive spot from the TLC plate showed that only a
small portion of the radioactivity in urine was intact
GPG-NH.sub.2. (See FIG. 7). The results from the in vivo studies
above proved that a significant amount of small peptides are
effectively delivered to blood, plasma, and several different
tissues.
[0172] Additionally, as shown in FIGS. 9 and 10, a significant
amount of small peptide remains in the plasma fraction over a long
period of time. In FIG. 9, the distribution of radioactivity
between blood cells, plasma protein bound, as well as, non protein
bound (free) plasma radioactivity is shown. The elimination of
radioactivity from the plasma fraction is depicted in FIG. 10. The
tripeptide GPG-NH.sub.2 has a half-life of 86.5 minutes. Protein
bound radioactivity was assayed after precipitation with two parts
of 99.5% ethanol. From the uptake and distribution data and the TLC
data above, the minimal uptake of intact GPG-NH.sub.2 recovered
from the plasma was calculated and, in one hour after feeding of
the rats, at least 1% of fed GPG-NH.sub.2 could be recovered as
protein free GPG-NH.sub.2 in the plasma.
[0173] For the assessment of biologically active GPG-NH.sub.2 in
the plasma of animals fed the small peptide, plasma samples were
prepared from blood obtained from the rats of the four week
toxicology study the day after the last feeding. The plasma samples
were diluted 1/5 in RPMI medium and were adminstered to PBMC
infected with the SF162 strain of HIV-1, as described above. Viral
infectivity was then monitored at seven, eleven, and fourteen days
post infection by detecting the amount of p24 in the supernatent
using a commercially available detection assay. (Abbott). As shown
in Table 12, the sera obtained from the rats treated with the small
peptide retained the ability to inhibit viral infectivity. In some
cases, the administration of as little as 10 .mu.M GPG-NH.sub.2
provided a sufficient concentration of small peptide in the plasma
to enable the inhibition of HIV-1 replication. The percent
reduction was calculated as in Table 6. The results from these
experiments established that small peptides described herein can be
maintained at concentrations in the body of an animal that are
effective at inhibiting HIV replication.
14 TABLE 13 Feeding GPG p24 % Animal #. (mg/ml) (pg/ml) reduction
Exp. 1 day 7 14 0 543.0 0 34 10 93.3 82.8 53 30 24.0 95.6 73 100
174.7 67.8 day 14 14 0 22678 0 34 10 1636 92.8 53 30 938 95.9 73
100 9211 59.4 Exp. 2 day 7 16 0 321.8 0 36 10 219.2 31.9 56 30
194.3 39.6 76 100 173.5 46.1 day 14 16 0 4075.4 0 36 10 4760.8 0 56
30 3574.4 12.3 76 100 2203.7 45.9 Exp. 3 day 18 0 183.9 0 38 10
255.6 0 58 30 107.3 41.7 78 100 96.9 47.3 day 14 18 0 7578.4 0 38
10 6700.6 11.6 58 30 6893.0 9.1 78 100 7578.4 0 Exp. 4 day 11 13 0
242 0 33 10 170 29.8 52 30 487.4 0 71 100 51.7 78.6 Exp. 5 day 7 15
0 304.8 0 35 10 79.6 73.9 55 30 439.3 0 75 100 60 80.3
[0174] The proteins from whole plasma were also analyzed by column
chromatography and fractionated and crude protein were separated by
sodium dodecyl sulfate polyacrylamide gel (10%) electrophoresis
(SDS/PAGE). Rat plasma samples were partially purified with size
exclusion (Sepharose G-50, Phamacia) chromatography (0.4.times.6
cm), in a buffer of 10 mM Tris-HCl pH 8.3 and 50 mM KCl. Eluate was
then separated by anionic exchange chromatography by using an
increasing stepwise gradient of NaCl in a buffer of 10 mM Tris-HCl
pH8.3 (Sepharose CL-6B DEAE, 0.4.times.6 cm). The eluate was
monitored and pooled according to the radioactivity (Ready Safe,
Beckman; LKB 1218, Sweden). The protein fractions with strong
signal were separated on a 10% SDS-PAGE and were subsequently
blotted onto PVDF (polyvinylidene difluoride, BioRad) membrane
before being subjected to Edman N-terminal amino acid sequencing
(Applied Biosystems Procise Sequencer, USA).
[0175] As shown FIG. 11, little radioactivity was observed to be
covalently associated with proteins 60 minutes after feeding the
animal. The proteins marked B1-B4 were sequenced and determined to
be alpha-1 antitrypsin (Bi), pentaxin (B-2), C-reactive protein
(B-3), and B-4 could not be identified. These proteins are all
synthesized in the liver. One interpretation is that hydrolyzed
GPG-NH.sub.2 was reutilized in the liver protein synthesis. These
experiments established that small peptides are metabolized in the
body and, since the identified associated protein (B-1, B-2, and
B-3) are all synthesized in the liver, hydrolysis and reutilization
of small peptides occured in the liver.
[0176] Although the invention has been described with reference to
certain embodiments and examples, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
* * * * *